chloroplast ribosomes and protein synthesis

MICROBIOLOGICAL REVIEWS, Dec. 1994, p. 700-754 Vol. 58, No. 40146-0749/94/$04.00+0Copyright © 1994, American Society for Microbiology

Chloroplast Ribosomes and Protein SynthesisELIZABETH H. HARRIS,`* JOHN E. BOYNTON,' AND NICHOLAS W. GILLHAM2

DCMB Group, Departments of Botany' and Zoology,2 Duke University, Durham, North Carolina 27708-1000

ORIGIN OF CHLOROPLASTS................................................................CHLOROPLAST GENOME STRUCTURE AND GENE CONTENT..THE PROCESS OF CHLOROPLAST PROTEIN SYNTHESIS...........

.700

..700

..702'rnlInitiation.....................................................................................................................................t.a..i.....n.... ... .....1....

-s - 17dkElongation.........4

F'0304

Chloroplast tRNAs and Aminoacyl-tRNA Synthetases..................................................................... 704PLASTID GENES FOR rRNAs ..................................................................... 704

Phylogenetic Conservation................................................... .................. 704General Characteristics of Chloroplast rRNA Gene Organization.........................................*70716S rRNA ..................................................................... 70723S rRNA.....................................................................7095S rRNA..................................................................o..709Introns in rRNA Genes ..................................................................... 709The 16S-23SSpacer .................................................................... 712tRNAs Flanking the rRNA Operons..................................................................... 712Antibiotic Resistance Mutations in the Chloroplast rRNA Genes......................1.....2.0..................712

RIBOSOMAL PROTEINS.......................................................... ........... 714Number and Nomenclature..................................................................... 714Organization of Chloroplast Ribosomal Protein Genes ....................................... ............*.................715Correspondence of Chloroplast Ribosomal Proteins to Bacterial Ribosomal Proteins................ ...............716Proteins of the Small Subunit.....................................................................716Proteins of the LargeSubunit............................................ .......................... 726Chloroplast Ribosomal Proteins with No Obvious Homology to Those of E. coli .................729Comparative Analysis of Ribosomal Proteins.......................................................30

ASSEMBLY OF CHLOROPLAST RIBOSOMES .....................................................................730SYNTHESIS OF THE COMPONENTS OF CHLOROPLAST RIBOSOMES .............................................., .. 731

Transcription of rRNA Genes..................................................................731Transcription of Chloroplast Genes Encoding Ribosomal Proteins .....................................................................732Posttranscriptional Regulatory Mechanisms Affecting Chloroplast mRNAs .......................... ..........733Membrane Binding of Chloroplast Ribosomes .....................................................................733

HOW ESSENTIAL IS CHLOROPLAST PROTEIN SYNTHESIS?...........-..................'............... ..........734CONCLUSIONS ..................................................................... 735ACKNOWLEDGMENTS.................... ....................... ...........................735REFERENCES....................................................I............"o..73

ORIGIN OF CHLOROPLASTS

Chloroplasts and mitochondria contain protein synthesizing-systems more similar to those of bacteria than to those of theeukaryotic cytoplasm, consistent with the hypothesis that theseorganelles had xenogenous (endosymbiotic) rather than autog-enous (intracellular differentiation) origins (see. references 5,205, 220-223, 274, 633, and 694 for discussions). Phylogeniesbased mostly on rRNA sequences indicate that the cyanobac-teria are ancestral to chloroplasts while the members of thealpha subdivision of the purple sulfur bacteria are the likelyprogenitors of mitochondria (221, 222). Whether the, chloro-phyte algae and land plants on the one hand, and the rhodo-phyte, chromophyte, and euglenoid algae on the other repre-

sent more than one endosymbiotic event remains unresolved(130, 403, 434). Comparisons of gene order and arrangement

* Corresponding author. Mailing address: DCMB, Duke UniversityBox 91000, Durham, NC 27708-1000. Phone: (919) 613-8164. Fax:(919) 613-8177. Electronic mail address: [email protected].

among these various taxa have produced intriguing directionsfor future evolutionary studies, while analysis of ribosomalprotein sequences, particularly among the diverse algal groups,promises. to be a valuable tool for determining conservedregions likely to have essential functions in ribosome assemblyor protein synthesis.

CHLOROPLAST GENOME STRUCTURE ANDGENE CONTENT

Unlike their prokaryotic ancestors, neither chloroplasts normitochondria are genetically autonomous, and informationspecifying components of the organelle protein synthesizingsystems is divided between organelle and nucleus. Separationof the genes encoding these RNAs and proteins between twodiscrete cellular compartments suggests that mechanisms musthave evolved to coordinate expression of these genes so thatprotein synthesis in the organelle can proceed efficiently.Whereas chloroplast genomes of land plants usually have acommon organization and gene content, a great deal morevariability is encountered among the algae, particularly with

700

A

CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 701

FIG. 1. Schematic diagram of a typical land plant chloroplastgenome (tobacco), showing the positions of the inverted repeat, rRNAgenes, and genes encoding ribosomal proteins. Gene locations are

from reference 560.

regard to the ribosomal protein genes that have been retainedin the organelle. In this section we review the chloroplastgenome structure of land plants and the algal genera that havebeen investigated to date with respect to composition andorganization of genes encoding rRNAs and ribosomal pro-

teins.Chloroplasts are highly polyploid organelles containing cir-

cular DNA molecules of 85 to 200 kb organized into discretemembrane-associated nucleoids (see references 50, 206, 260,337, 482, 483, 558, 613, and 614 for reviews). Three land plantchloroplast genomes have been completely sequenced: thedicotyledon tobacco (Nicotiana tabacum, 156 kb [560, 561]),the monocotyledon rice (Oryza sativa, 135 kb [265]); and a

liverwort (Marchantia polymorpha, 121 kb [471-473]). Eachcontains 110 to 120 genes (482, 614). These sequences, as wellas restriction maps and partial sequences from many otherspecies, indicate that the basic chloroplast genome structureand overall gene order in land plants are highly conserved.Although green algae (Chlorophyta) are regarded as ancestralto land plants, modern green algae often show substantialrearrangements in chloroplast gene order (see below). Othergroups of algae (Rhodophyta, Euglenophyta, Chromophyta)show even more diversity in gene content and organization.

In the typical land plant chloroplast genome, unique se-

quence regions of 15 to 25 kb and 80 to 100 kb are separatedby the two copies of an inverted repeat, which is usually 20 to30 kb in size and contains genes encoding the chloroplastrRNAs, certain tRNAs, and often one or more genes specify-ing proteins (Fig. 1) (see references 482 and 614 for reviews).Within the inverted repeat, the rRNA operon is usuallyoriented with the 23S rRNA gene closer to the small single-copy region and the 16S rRNA gene closer to the largesingle-copy region. The two repeats are identical in sequence

as a consequence of an active copy correction system (50).Nearly two-thirds of the variation in size among land plantchloroplast genomes (120 to 216 kb) is accounted for byexpansion or contraction of the inverted repeat (482). Thesmallest chloroplast genomes among land plants are seen inconifers (355, 600, 695) and in six tribes of the legume family

Fabaceae (314, 482, 487), which have lost the inverted repeatand thus contain only a single copy of each of the rRNA genes.Black pine (Pinus thunbergii) chloroplast DNA does possess ashort inverted repeat sequence, which contains a tRNA geneand part of the 3' portion of the psbA gene, but not the rRNAgenes (654). In contrast, species with the largest chloroplastgenomes often have expanded inverted repeats (e.g., Pelargo-nium hortorum has a 76-kb inverted repeat encompassingnearly half of the 216-kb chloroplast genome, in which manygenes normally in the single-copy region have been duplicated[482]).

Chloroplast genomes from land plants specify a relativelyconstant set of components for the protein-synthesizing ma-chinery of the organelle (4 rRNAs, 30 to 31 tRNAs, 21ribosomal proteins, and 4 RNA polymerase subunits) and forphotosynthesis (28 thylakoid proteins plus 1 soluble protein,the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco]large subunit). In addition, homologs of 11 subunits of mam-malian mitochondrial complex I (the ndh genes) have nowbeen found to be encoded by chloroplast DNA in floweringplants and Marchantia species (9,713). Chloroplast genomes ofgymnosperms, liverworts, and algae (e.g., Chlamydomonasreinhardtii) which synthesize chlorophyll in darkness possessgenes encoding three subunits of a light-independent proto-chlorophyllide reductase that is also found in photosyntheticprokaryotes (see reference 367 for a summary). These genesare absent from the tobacco and rice chloroplast genomes.Mapping and sequencing studies of chloroplast genomes

from widely different algal taxa reveal that these are muchmore variable in organization and gene content than those ofland plants. The well-characterized chloroplast genomes ofthree species of unicellular green algae in the genus Chlamyd-omonas are substantially larger (C. reinhardtii, 196 kb; C.eugametos, 243 kb; C. moewusii, 292 kb) than the chloroplastgenomes of land plants (42, 43, 50, 247). In these species thetwo copies of the large inverted repeat encoding the rRNAsare separated by unique sequence regions of roughly equalsize. Chloroplast genes in Chlamydomonas species are alsoextensively rearranged between distantly related species andwith respect to land plants (43). The green alga Spirogyramaxima, in the charophyte lineage presumed to be ancestral toland plants, lacks an inverted repeat and shows altered geneorder relative to land plants (352, 393).The organization, structure, and gene content of the com-

pletely sequenced 145-kb chloroplast genome of Euglena gra-cilis Z (243) depart markedly from the chloroplast genomes ofchlorophyte algae or land plants. In this Euglena strain and inits colorless relative Astasia longa, the plastid genome containsthree tandemly repeated rRNA operons plus an additional 16Sgene or fragment thereof (288, 289, 315-317, 478, 569).Euglena gracilis var. bacillaris has only a single complete rRNAoperon (720). Most of the Euglena chloroplast tRNA genes aregrouped in tight clusters of two to five genes, whereas they tendto be scattered in plastid genomes of land plants. While mostprotein-coding chloroplast genes in land plants or Chlamydo-monas species are uninterrupted or contain at most one or twointrons, comparable genes in Euglena gracilis each containmultiple introns (243, 482). However, several chloroplasttRNA genes that have introns in land plants lack introns inEuglena or Chlamydomonas species (331). A number of othergenes found in land plant chloroplast genomes, including threegenes encoding ribosomal proteins, are missing from theEuglena chloroplast genome (see below), but this algal genomealso contains some genes not found in plastid genomes of landplants.The plastid of Cyanophora paradoxa is often referred to as

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the cyanelle because of its secondary peptidoglycan wall andphotosynthetic apparatus with phycobiliproteins typical of cya-nobacteria and red algae. Most of the 133-kb cyanelle genomehas now been sequenced (32, 598). This genome contains aninverted repeat which encodes the cyanelle rRNAs and severalother genes. Although the gene content of the cyanelle gener-ally resembles that of land plant chloroplasts, there are about30% more genes, including 11 additional genes encodingcomponents of the translational apparatus. So far, only a singletype I intron has been found in Cyanophora paradoxa, in atRNAIeU gene (162). The same intron is found in cyanobacte-ria.Cryptomonad algae contain a plastid and residual nucleus or

nucleomorph enclosed within the endoplasmic reticulum of thecytoplasm and thus effectively separated from the normal cellnucleus (see 123, 386). Distinctly different 18S rRNAs areencoded by the nucleus and nucleomorph of Cryptomonas (Dand are spatially separated within the cell (129, 414). Thenucleomorph rRNA genes are related to those of red algae,while the nuclear rRNA genes are clustered separately in thephylogenetic branch containing land plants and green algae.This suggests that cryptomonad algae may have arisen througha second endosymbiotic event in which a eukaryotic symbiontfrom the red algal lineage was taken up by a unicellular hostmore closely related to the green algae (129, 130, 386). Partialsequencing of the plastid genome of Cryptomonas (D hasrevealed the presence of several novel genes, including fourgenes for ribosomal proteins not found in chloroplast genomesof land plants (122, 124, 680; also see below).

In the red alga Porphyra purpurea, over 125 genes have beenidentified in the ca. 60% sequenced chloroplast genome (514,515), suggesting that the entire genome may contain as manyas 200 to 220 genes, about twice as many as found in thecompletely sequenced genomes of land plant chloroplasts.These include at least seven photosynthesis and nine ribosomalprotein genes not present in land plants. Introns have not beenfound in any of the 80 genes sequenced to date. The chloro-plast genome of P. yezoensis possesses an inverted repeatcontaining the rRNA genes (353, 562, 563), but the related redalgae P. purpurea and Grijflthsia pacifica lack this inverted-repeat structure. In P. purpurea the rRNA genes are encodedin direct repeats which are not identical in sequence (514, 516,563). The unicellular red alga Cyanidium caldarium possessesan inverted repeat containing only the rRNA genes, but geneorder appears to be more similar overall to that of Cryptomo-nas (D than to that of P. yezoensis or Griffithsia pacifica (385).

Inverted repeats containing rRNA genes are also found inthe plastid genomes of the brown alga Dictyota dichotoma(330) and the golden-brown algae Olisthodiscus luteus andOchromonas danica (108, 563). The plastid genome of thebrown alga Pylaiella littoralis contains two different circularDNA molecules (369, 370, 404, 405). The larger (133 kb)molecule resembles a typical land plant chloroplast genome,with two rRNA operons in an inverted repeat. The smaller (58kb) molecule contains a 16S pseudogene sequence, which is65% homologous to the functional 16S genes of the largemolecule, and a complex region that hybridizes with a 23SrRNA probe (369, 370).

THE PROCESS OF CHLOROPLASTPROTEIN SYNTHESIS

We begin this brief review of chloroplast protein synthesiswith a comparison with the process as it occurs in bacteria. Thissection will be followed by discussion of the tRNAs andaminoacyl-tRNA synthetases. A more detailed discussion of

chloroplast protein synthesis has been presented by Steinmetzand Weil (593).

Initiation

In prokaryotes, protein synthesis begins with formation of apreinitiation complex from the 30S ribosomal subunit andtRNAIMetUAC, with the 30S subunit binding to the purine-richShine-Dalgarno sequence 7 ± 2 nucleotides (nt) upstream ofthe initiator AUG (230, 261, 323). The canonical Shine-Dalgarno sequence, GGAGG, or a variant, pairs with apyrimidine-rich complementary sequence, the anti-Shine-Dal-garno sequence, near the 3' end of the 16S rRNA molecule.Addition of a50S ribosomal subunit converts the preinitiationcomplex to an initiation complex that can enter the elongationphase of protein synthesis. These reactions are promoted bythe three initiation factors, IF-1, IF-2, and IF-3. IF-1 enhancesthe rates of ribosome dissociation and association and theactivities of the other initiation factors (261). IF-2 is involved ininitiator tRNA binding and GTP hydrolysis, while IF-3 pre-vents ribosomal subunit association in the absence of mRNAand appears to stabilize mRNA binding by promoting theconversion of a preternary ribosome-mRNA-fMet tRNA com-plex into a ternary complex in which codon-anticodon interac-tion has occurred. IF-3 also is thought to proofread theAUG-anticodon interaction. Chloroplast equivalents of IF-2and IF-3, designated IF-2Ch, and IF-3Chl, have been character-ized from Euglena gracilis (212, 324, 325, 375,527, 678). Roneyet al. 527) confirmed that IF-2Chl is required for binding oftRNA et to chloroplast 30S subunits, as is prokaryotic IF-2.IF-2,hl occurs in several complex forms, varying in molecularmass from 200 to 800 kDa (375). Subunits of 97 to >200 kDahave been observed in these preparations. IF-3,hl promotesinitiation complex formation in the presence of IF-2Chi. Al-though IF-3Chl will replace Escherichia coli IF-3 in initiationcomplex formation, there is some evidence that its functionmay be modified (527).A DNA sequence with homology to the E. coli infA gene

encoding initiation factor IF-1 has been identified in thechloroplast genomes of land plants, including the colorlessparasite Epifagus virginiana (558, 714), but is apparently absentfrom the completely sequenced chloroplast genome of Euglenagracilis (243). The tobacco infA gene, in contrast to the spinachgene (571), lacks the ATG translation initiation codon andthus may be a pseudogene. Reading frames with homology tothe genes encoding IF-2 and IF-3 have not been detected in thesequenced plastid genomes of green plants, Epifagus virginiana,or Euglena gracilis, and inhibitor experiments suggest that theEuglena genes specifying these factors are nuclear in location.However, homologs of the infB gene encoding IF-2 have beenfound in the chloroplast genomes of the red algae P. purpurea(514) and Galdieria sulphuraria (322). Lin et al. (359) haverecently reported characterization of a cDNA clone encodingIF-3Chl in Euglena gracilis. This nuclear gene appears to bepresent in about four copies, one of which is probably apseudogene. The putative protein contains two acidic regionswith no homology to other known sequences, in addition to a175-amino-acid region with 31 to 37% homology to other IF-3proteins.

Shine-Dalgarno-like sequences are present in the untrans-lated leader regions of many but not all chloroplast mRNAs(35, 44, 318, 532, 593, 746). Ruf and Kossel (532) reported that37 of 41 chloroplast genes examined in tobacco have suchsequences if one extends the anti-Shine-Dalgarno sequence inthe 16S rRNA beyond the canonical CCUCC to include theadjacent unpaired ACUAG sequence. Bonham-Smith and

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Bourque (35) observed that 181 of 196 chloroplast-encodedtranscripts examined possessed a Shine-Dalgarno sequencewithin 100 bp 5' to the initiation codon. However, spacing ofShine-Dalgarno sequences in chloroplast mRNAs is less uni-form than in bacteria. Frequency distributions of the mostcommon individual positions potentially involved in base pair-ing with 16S rRNA ranged from -2 to -29, with a major peak(ca. 40%) at -7 to -8, a smaller peak at -15 to -16, and athird small peak at -21 to -23 (35, 532). Thus, chloroplastribosomes may be able to accommodate larger distancesbetween the ribosome recognition site and translational startsites than bacterial ribosomes. For example, in the Chlamydo-monas rpsl2 gene, a canonical Shine-Dalgarno sequence isfound at position -55 upstream of the initiator codon (364).The variability of the Shine-Dalgarno sequence raises thequestion whether initiation from this sequence proceeds as ineubacteria for Shine-Dalgarno sequences close to the AUGcodon and occurs by transient binding and "scanning" formore-distant Shine-Dalgarno sequences (306). In tobacco themRNAs for those chloroplast genes lacking Shine-Dalgarnosequences either show only a trinucleotide sequence for po-tential base pairing (atpB) or contain out-of-frame initiatorcodons between the potential recognition sites and the respec-tive in-frame start codons (rpsl6, rpoB, and petD [532]).

In Euglena chloroplasts, mRNA-rRNA recognition seems toproceed by somewhat different rules, because the putativeanti-Shine-Dalgarno sequence CUCCC differs from the canon-ical CCUCC sequence and actually forms the 3' terminus ofthe 16S rRNA rather than being located several bases from theend (592). Since only about half of the Euglena chloroplastmRNAs contain Shine-Dalgarno sequences, two modes ofinitiation complex formation have been postulated (527, 677).In one class of mRNAs, complex formation is facilitated by aShine-Dalgarno-like sequence. However, in the second classthe A+U content of the region 5' to the initiator AUG is 90%or greater and this portion of the mRNA is relatively unstruc-tured, making potential start sites in this region readily acces-sible to small subunits. Koo and Spremulli (318, 319) havestudied formation of initiation complexes in vitro with tran-scripts containing the 5' untranslated leader region of theEuglena rbcL mRNA, which is A+U rich and contains noShine-Dalgarno sequence. Introducing a Shine-Dalgarno se-quence into this region enhanced initiation only slightly.Deletion and/or modification of the leader region demon-strated that a minimum of about 20 nt is required to form theinitiation complex in vitro and that the full 55-nt length isnecessary for full activity in complex formation (318). Theprimary sequence of the region seems less important forinitiation than does its length. The native 55-nt sequence hasonly weak secondary structure, and modification of the se-quence to create increased secondary structure within about 10nt of the AUG codon diminished formation of the initiationcomplex significantly (319). Koo and Spremulli concluded thatthe major determinant of initiation in those Euglena mRNAswith no Shine-Dalgarno sequence is presence of the AUGcodon in an unstructured region ofmRNA that is accessible tothe 30S subunit.

Elongation

Elongation of the peptide chain requires three steps, i.e.,aminoacyl-tRNA binding, peptide bond formation, and trans-location, and involves three binding sites for tRNA (261, 460,518, 690). The aminoacylated tRNA combines with elongationfactor EF-Tu and GTP to form a ternary complex, which thenassociates with a ribosome complexed to mRNA and peptidyl-

tRNA. The specific ternary complex is selected on the basis ofcodon-anticodon recognition at the A site and is followed byGTP hydrolysis and the release of an EF-Tu-GDP complex.Peptide bond formation takes place with transfer of thegrowing peptide chain to the aminoacyl-tRNA in the A site.Translocation is promoted by EF-G and GTP hydrolysis, andinvolves movement of the peptidyl-tRNA-mRNA complexfrom the A to the P site. The process is then repeated, and thedeacylated tRNA moves from the P to the E site. The A and Esites themselves are allosterically linked in a negative sense sothat occupation of the A site by aminoacylated tRNA reducesthe affinity of the E site for deacylated tRNA and vice versa.Regeneration of the active EF-Tu-GTP complex from EF-Tu-GDP is mediated by elongation factor EF-Ts. All three elon-gation factors have been characterized from Euglena chloro-plasts by Spremulli and colleagues (53, 145, 173, 341, 585), andthe structure of the guanine nucleotide-binding domain ofEF-Tu has been modeled by Lapadat et al. (341). EF-Tu hasalso been purified from pea and tobacco chloroplasts (445,589).

Reading frames with homology to the bacterial genes en-coding the three elongation factors EF-Tu, EF-G, and EF-Tsare absent from the three completely sequenced land plantchloroplast genomes (482, 558), but some of these genes havebeen retained in the plastid genomes of certain algae (seebelow). Two distinct nuclear genes encoding chloroplastEF-Tu have been identified in tobacco (445, 611, 661). Anuclear EF-G gene has been cloned and sequenced fromsoybean (650), and a partial clone obtained from pea (2).

Early inhibitor experiments with Euglena gracilis indicatedthat EF-Ts and EF-G were nuclear gene products but thatEF-Tu might be encoded in the chloroplast (52, 173). Thesepredictions were confirmed by identification of a chloroplasttufA gene encoding EF-Tu (429) and by failure to find genesencoding EF-Ts or EF-G in the recently completed Euglenachloroplast genome sequence (243). The Euglena tufA gene issplit into three exons separated by two introns (429). Anuninterrupted sequence with homology to the E. coli tufA genehas been reported from the chloroplast genome of C. rein-hardtii (15, 684). The tufA gene sequence is also found in theCyanophora cyanelle genome (32, 598) and in the chloroplastgenomes of representative green algae in the families Ulvo-phyceae, Chlorophyceae, and Charophyceae, the latter groupbeing the presumed ancestors of land plants (14, 15). However,tufA is absent from the chloroplast genome of the liverwortMarchantia polymorpha, representative of the earlier land plantlineages (472, 473). Baldauf and Palmer (15) concluded thattransfer of this gene to the nucleus probably occurred in thecharophycean lineage prior to the emergence of land plants.

Reith and Munholland (514) have reported that the chloro-plast genome of the red alga P. purpurea not only possesses areading frame corresponding to tufA but also possesses onecorresponding to tsfwhich encodes EF-Ts in prokaryotes. Thisgene has also been found in the chloroplast genome of thethermophilic red alga Galdieria sulphuraria (322). In pro-karyotes, mutations to fusidic acid resistance can occur in thestructural gene for EF-G (fus) (357). A nuclear mutation in C.reinhardtii has been reported to confer fusidic acid resistanceon chloroplast EF-G, but the gene encoding this factor has notyet been identified (74; also see 247).

Production of chloroplast protein synthesis factors appearsto be light regulated. Spremulli and coworkers have shown thatactivities of Euglena IF-2, IF-3, EF-Tu, EF-G, and EF-Ts allincrease on transfer of cells from dark growth to light (52, 173,324, 585). In Chlamydomonas synchronous cultures, transcrip-tion rates for four chloroplast-encoded photosynthetic genes

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and for the tufA gene were all found to be maximal at thebeginning of the light period (51, 350). However, EF-TumRNA decreased to almost undetectable levels in the secondhalf of the light period. Activity of the pea chloroplast EF-G,encoded by a nuclear gene, is also light regulated but at thelevel of translation (1).

Termination

Termination of translation in bacteria involves the hydrolysisof peptidyl-tRNA and release of the completed protein fromthe ribosome when the ribosome reaches one of the threetermination codons (261). Termination requires the action oftwo release factors, RF-1, which is specific for UAA and UAG,and RF-2, which is specific for UAA and UGA. A third releasefactor, RF-3, stimulates the activities of RF-1 and RF-2. Thesame three codons are used for translation termination inchloroplasts (35, 36), with UAA being by far the most frequent(70% in land plant sequences surveyed by Bonham-Smith andBourque [36]) and UGA being rare (9%). UAA is alsooverwhelmingly preferred as the stop codon in Chlamydomo-nas chloroplast genes (247). Bonham-Smith and Bourque (35)noted that UGA was never used as a stop codon in Marchantiachloroplast genes and proposed that a modification of the 16SrRNA in this species prevents recognition of UGA as atermination signal. No reading frame with homology to any ofthe genes encoding bacterial termination factors has beenidentified in a chloroplast genome, nor has isolation of thesefactors been reported.

Chloroplast tRNAs and Aminoacyl-tRNA Synthetases

The properties of chloroplast aminoacyl-tRNA synthetaseshave been summarized by Steinmetz and Weil (593). Theseenzymes are encoded in the nucleus. Most are distinguishablefrom their cytoplasmic counterparts and will charge onlychloroplast or prokaryotic tRNAs efficiently. These enzymeshave unusually high molecular masses (75 kDa or greater) andcan be found as monomers, homodimers, heterodimers, orheterotetramers depending on the enzyme.The structure and codon recognition patterns of chloroplast

tRNAs and the organization of their cognate genes have beenextensively reviewed elsewhere (397-399, 593, 616). Genesencoding individual chloroplast tRNAs are highly conserved indifferent species of land plants and are similar in structure andsequence (ca. 70% sequence identity) to prokaryotic tRNAgenes but have low homology to those of eukaryotic cells.However, the 3'-terminal CCA triplets of chloroplast tRNAsare added posttranscriptionally, as occurs for all eukaryoticcytoplasmic tRNAs but for only about one-third of bacterialtRNAs. Isoaccepting tRNAs for a given amino acid areencoded by different chloroplast genes, but these tRNAs arecharged by the same chloroplast tRNA synthetases. Somechloroplast tRNA genes are preceded by prokaxyotic-likepromoter sequences, but such sequences are absent upstreamof other chloroplast tRNA genes, which may thus possessalternative promoters, possibly internal to the coding region(227, 229, 616).The tobacco chloroplast genome contains 30 tRNA genes,

23 of which are single and 7 of which are duplicated in theinverted repeat. Rice has the same set of tRNA genes astobacco, but the inverted repeat extends through the tRNAH1Sgene, found in the single-copy region adjacent to the invertedrepeat in tobacco. In liverwort there are 31 chloroplast-encoded tRNA genes, with the extra gene being tRNAAxgCCG,but in Euglena gracilis there are only 27 (243, 558, 616).

Several chloroplast tRNAs have unusual features. TIwodifferent tRNAIle species are found in plant chloroplasts. Themajor species (tRNAIle1, encoded in the spacer between the16S and 23S genes) recognizes the codons AUU and AUC,while a minor species (tRNAIle2) recognizes AUA. However,the gene encoding the latter tRNA contains a CAU anticodon,which normally would recognize AUG for methionine. Onepossible explanation is that the C residue is modified in someway posttranscriptionally. In E. coli the C of the homologoustRNA is modified to lysidine, a novel type of cytidine with alysine residue, which allows it to recognize the AUA codon(444).One tRNAGlUuc has a special function in chlorophyll

biosynthesis as well as participating in protein synthesis, whilethe other two species have a U*UG anticodon specific forglutamine and are converted from Glu-tRNA01n to Gln-tRNAGJn by a specific amidotransferase activity present inchloroplast extracts (398, 616). This mischarging mechanismhas also been described in several gram-positive bacteria (398).The chloroplast genomes sequenced to date encode a typical

initiator tRNA"cICAu, and all employ the three classicaltermination codons (UAA, UAG, and UGA). However, genesfor tRNAs recognizing the codons CUU/C (Leu), CCU/C(Pro), GCU/C (Ala), and CGC/A/G (Arg) are absent from thechloroplast genomes of tobacco and rice. Since all 61 sensecodons are used in the three sequenced land plant chloroplastgenomes, this deficit in specific tRNAs requires that thetRNAs either be imported or be read by the "two-of-three"mechanism used in animal mitochondria (174, 716) or byfour-way wobble (480). In the absence of import in Euglenachloroplasts, one of the last two mechanisms would have topertain to seven of the eight codon families (243). In land plantchloroplasts, two-of-three or four-way wobble seems to be usedfor tRNAMIaUda,GC, tRNAProU*GC, and tRNAA`gICG, which canread respectively all four alanine (GCN), proline (CCN), andarginine (CGN) codons (488). The first two tRNAs contain amodified U (U*) in the anticodon. The problem of decodingthe six leucine codons is solved somewhat differently. Two ofthe leucyl-tRNAs translate the UUA and UUG codons (488).The remaining tRNAeUUAM7G translates all four CUNcodons for leucine apparently by a U * N wobble mechanism(489).

In tobacco, rice, and liverwort, six of the chloroplast-en-coded tRNA genes possess introns which must be removedfrom the primary transcript during processing (398). In to-bacco these introns range in size from 503 bp (tRNAeuuAA)to 2,526 bp (tRNALYSJuu) (616). Many land plant chloroplasttRNAs are singly transcribed, although a cotranscribed, tricis-tronic tRNA gene cluster has been identified in tobacco (398)and the two tRNAs found in the spacer between the 16S and23S rRNA genes are transcribed as part of the rRNA operonprecursor (see below). Cotranscription of tRNA gene operonsis the usual case in Euglena gracilis. RNase activities thought tobe involved specifically in tRNA processing have been identi-fied in chloroplast extracts (225, 226, 727).

PLASTID GENES FOR rRNAs

Phylogenetic ConservationAll chloroplast genomes examined contain genes for the

16S, 23S, and 5S RNAs of the chloroplast ribosome. Table 1lists species for which sequences have been published. Chlo-roplast rRNAs are highly conserved at the sequence level andare most closely related to eubacterial sequences, which in-clude those of cyanobacteria (210, 219, 236, 512, 709). For

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TABLE 1. Plastid and cyanobacterial rRNA sequences published or submitted to GenBank

Taxon GenBank accession no(s). Reference(s)

16S rRNAAnabaena sp.Anacystis nidulansAntithamnion sp.Astasia longaChlamydomonas moewusiiChlamydomonas reinhardtiiChlorella ellipsoideaChlorella kessleriChlorella mirabilisChlorella protothecoidesChlorella sorokinianaChlorella vulgarisConopholis americanaCryptomonas 1Cyanidium caldariumCyanobacteria (miscellaneous spp.)

Cyanophora paradoxaDaucus carotaEpifagus virginianaEuglena gracilisEuglena gracilis bacillarisGlycine maxHelianthus annuusMarchantia polymorphaNanochlorum eucaryotumNicotiana plumbaginifoliaNicotiana tabacumOchromonas danicaOchrosphaera sp.Olisthodiscus luteusOryza sativaOscillatoria sp.Palmaria palmataPisum sativum

Porphyra purpureaPorphyridium sp.Prochloron sp.Pylaiella littoralisPyrenomonas salinaSinapis albaSpinacia oleraceaSpirodela oligorhizaSynechococcus lividusZea mays

23S rRNAAlnus incanaAnacystis nidulansAntihamnion sp.Astasia longaChlamydomonas eugametosChlamydomonas frankiiChlamydomonas gelatinosaChlamydomonas geitleriChlamydomonas humicolaChlamydomonas indicaChlamydomonas iyengariiChlamydomonas kommaChlamydomonas mexicanaChlamydomonas moewusiiChlamydomonas pallidostigmaticaChlamydomonas peterfiiChlamydomonas pitschmanii

X59559X03538; X00346, K01983 (partial)X54299X14386X15850J01395, X03269X12742, X05694, X03848X65099X65100X65688X65689X16579X58864X56806X52985M63813, M63814M62775, M62776M64522, M64526, M64531, M64536M19493 (partial)X73670M81884, X62099V00159, X12890, X05005, X70810X00536 (partial)X07675, X06428, M37149 (partial)X73893X04465X76084M82900, X70938J01452, J01453, V00165, V00166, Z00044X53183X65101M82860, X15768X15901X58359, X58360, X58361 (partial)Z18289M37430X51598M16874, M16862, M30826 (partial)L07257, L07258

X63141M21373, X14873, X14874X55015M15915, X04182J01440, M21453 (partial)X00014, X00015 (partial)X67091, X67092, X67093 (partial)M10720, Z00028

M75722X00512, X00343 (partial)X54299 (partial)X14386Z17234X68905-X68909Z15151X68891, X68892X68921, X68922X68893-X68898X68886, X68886X68927-X68929X68910-X68912X68913-X68918X68899-X68904X68887, X68888Z15152

356333, 647, 697384569140137722, 724, 725281281280280279702, 70313038363681-683, 692548287395435, 712, 714217, 243, 529, 530, 549152110, 67377312, 472, 473553476, 729560, 561, 645, 646705281108, 109265698, 69957360279557, 617, 61851634555, 660, 665404, 40538250256, 40930238554

351126, 334384569200, 657, 658658658658658658658658658658658658658

Continued on following page

VOL. 58, 1994

706 HARRIS ET AL.

Taxon

Chlamydomonas reinhardtiiChlamydomonas starriiChlamydomonas zebraChlamydomonas sp.Chlorella ellipsoideaColeochaete orbicularisConopholis americanaCryptomonas (FCyanidium caldariumCyanophora paradoxaEpifagus virginianaEuglena gracilisMarchantia polymorphaNicotiana tabacumOlisthodiscus luteusOryza sativaPalmaria palmataPisum sativumPylaiella littoralisSpinacia oleraceaSpirodela oligorhizaZea mays

4.5S rRNAAcorus calamusAllium tuberosumAlnus incanaApium graveolusCodium fragileCommelia communisConopholis americanaDryopteris acuminataGossypium hirsutumHordeum vulgareJungermannia subulataLigularia calthifoliaLycopersicon esculentumMarchantia polymorphaMarsilia quadrifoliaMnium rugicumNicotiana tabacumOryza sativaOsmunda regalisPisum sativumSpinacia oleraceaSpirodela oligorhizaTriticum aestivumZea mays

5S rRNAAlnus incanaAnacystis nidulansAstasia longaChlamydomonas reinhardtiiChlorella ellipsoideaConopholis americanaCyanophora paradoxaCycas revolutaDryopteris acuminataEuglena gracilis bacillarisEuglena gracilisGinkgo bilobaGlycine maxGossypium hirsutumJungermannia subulataJuniperus mediaLemna minorLupinus albusMarchantia polymorpha

TABLE 1-Continued

GenBank accession no(s).

J01398, X01977, X16687, X16686X68889, X68890X68919, X68920X68923-X68926M36158; X05693, X03848 (partial)X52737 (partial)X59768X14504 (partial)X54300 (partial)M19493 (partial)M81884, X62099X13310, X12890M13809, X04465, X01647J01446, Z00044X15768 (partial)X15901Z18289M37430X61179, M21373 (partial)M21453, X04977 (partial)X00012, X00013 (partial)Z00028, X01365

M36166M35406M75719M35404M35276M35407X58863X01523X63124M35405, M57605M13808M36165M33098X04465, M13809X51641M35056J01446, V00161, J01891, J01451, X01277, Z00044X15901X51978M37430M10757, X04977J01439M10541M19943, Z00028, X01365

M75719X00343, X00757, M23834X14386X03271X04978X58863M32451, M33030X12787X00758X00536K02483, X12890X51979X16736X63124X00667

X02714X65030X00666, X04465

Reference(s)

346, 521658658658726394703128384287435, 712, 714549, 730312, 473560, 561, 627108265573602405, 58111, 409304148

3173828473817673870362344080, 73869131739472, 473, 691421652560, 561, 624, 62526542160211, 332303696147, 148, 601

28494, 125569550724703411, 412743629152243, 29642123440728663144327472, 473, 728


MICROBIOL. REV.


TABLE 1-Continued

Taxon GenBank accession no(s). Reference(s)

Nicotiana tabacum J01451, M10360, M15995, X01277, Z00044 144, 560, 561, 624-626Oryza sativa X15901 265Picea excelsa X63200 421Pelargonium zonale X05551 146Pisum sativum M37430 602Porphyra purpurea L07259, L07260 516Porphyra umbilicalis 664Prochloron sp. K03159, X02637 380Pylaiella littoralis X61179 580Spinacia oleracea V00169, X05876 112, 491, 492Spirodela oligorhiza J01439 303Synechococcus lividus X02731 111, 113Vicia faba 663Zea mays M19943, Z00028 147, 601

example, primary sequence homology is generally over 70%for chloroplast or cyanobacterial 16S rRNAs compared withthat of E. coli and greater than 80% for chloroplast 16S rRNAcompared with cyanobacterial 16S rRNAs. Gray (219) recog-

nized eight noncontiguous conserved primary sequences in 16SrRNA interspersed among nonconserved sequences. The pre-

dicted secondary structures of these molecules are even more

conserved, and virtually all of the approximately 45 helicespostulated for the E. coli 16S rRNA (62, 462) are present inchloroplast 16S rRNAs of Euglena gracilis, Chlamydomonasspecies, tobacco, and maize (232, 512; also see below). Com-pensating base substitutions are often seen on the complemen-tary sides of predicted stem structures, strengthening thesupposition that these structures are functional in vivo. Be-cause of this high degree of structural conservation, rRNAgenes have found extensive use in phylogenetic studies (78,219, 232, 235, 236, 342, 710). Comparative analyses of 16S (54,210) and 5S (144, 380, 663, 743) rRNA sequences support boththe probable origin of chloroplasts from endosymbiotic cya-nobacteria and the hypothesis that land plants derive from onebranch of chlorophyte algae. Van de Peer et al. (665) havecompared 16S and 18S sequences from eukaryotic, archaebac-terial, eubacterial, plastid, and mitochondrial ribosomes. Al-though their analysis focused largely on mitochondrial origins,their data also support the common ancestry of cyanobacteriaand plastids.

General Characteristics of Chloroplast rRNAGene Organization

As in the eubacteria, chloroplast rRNA genes are normallyarranged in an operon transcribed in the order 16S-23S-5S(Fig. 2) (114, 320). In land plants, including some but not allferns, approximately 95 nt homologous to the 3' terminus ofthe E. coli 23S molecule constitutes a 4.5S rRNA molecule,separated from the remainder of the 23S gene by a transcribedspacer, whereas in prokaryotes, all algae so far examined,mosses, and the liverwort Marchantia polymorpha, the equiva-lent sequence is part of the 23S gene (47, 320). In C. reinhardtii,the sequences homologous to the 5' portion of the 23S gene ofbacteria and plants are divided into 7S and 3S rRNAs,separated by short spacers that are removed from the precur-sor rRNA posttranscriptionally (137). The large subunit rRNAof C. eugametos comprises species (a and ,B) equivalent to theC. reinhardtii 7S and 3S rRNAs and two larger species (ry and 8)which together are equivalent to the remainder of the 23Smolecule (656).

16S rRNA

The secondary-structure model of 16S rRNA based oncomparative sequence analysis (231, 232, 236, 449, 463, 468)suggests a functional division into distinct 5', central, and 3'domains, corresponding in E. coli to residues 26 to 557, 564 to912, and 926 to 1391, respectively, followed by a "3' minordomain" from ca. 1401 to 1542 (Fig. 3; for a numbered E. colisequence diagram in similar format to the tobacco sequenceshown in Fig. 3, see references 231 and 235). Each of thesedomains comprises helices and loops whose secondary struc-ture is phylogenetically conserved (219, 236). Models for thetertiary structure of the E. coli 30S subunit have been con-structed based on studies of RNA-RNA and RNA-proteincross-linking, immunoelectron microscopy, and neutron dif-fraction (58-61, 463, 465, 596). Functional analyses involvingmutants, binding of tRNA and antibiotics, and assembly ofribosomal proteins with RNA in vitro indicate that codon-anticodon recognition involves the 3' domain and terminal 3'minor domain. Three regions of the 16S molecule (E. coli nt518 to 533, 1394 to 1408, and 1492 to 1505) that show aparticularly high degree of primary sequence conservationappear to have tertiary interactions related to decoding (468).tRNA bound in the A site interacts specifically with the 3'domain and with residues in the "530 loop" (see reference 465for review), whereas P-site-bound tRNA protects five sites inthe central and 3' domains that are proposed to be clustered in

tRNA168 lie Al 23S 5s

_uI IEtRNA

cyanobacteria, most algae

165 I 23S 4.5S 5S

land plantstRNA

165 IleAla 7S 38 23S 5' 23S 3' 5S

C. relnhardtll

FIG. 2. Arrangement of the rRNA operons in land plants andalgae, showing conservation of tRNAIle and tRNAMa within the spacerbetween the 16S and 23S genes and variation in the species thatconstitute the 23S molecule. In land plants, the tRNA genes are splitby introns, whereas in all algae examined to date they are uninter-rupted. The region corresponding to the 3' end of the eubacterial 23SrRNA is a separate 4.5S rRNA in angiosperms, gymnosperms, andsome (but not all) ferns. Internal transcribed sequences and one ormore introns interrupt the 23S genes of Chlamydomonas species (658).

VOL. 58, 1994

708 HARRIS ET AL. MICROBIOL. REV.

central domainUA GA

a^o a CMnGAA A c^^cAA aGUc 'aGGUGGCCUUUAAGGG-cCA CGA

A Ua-CA Uc -aa0-C

GO 0U.GG-CC - GA

U - AC0-CA-U

ACCCACA AA cc U AAACCCUG0 GGCGOUGGA CU A AAGCC 11I1II1I1 I .II I .II II IIIIIlIi CACUCGOGACC CUGCCGCCU GA UUUUUC GU/.UAG

CU A A 0. A0!UAA G~~~~~A A

helix 17

5' domain

isr

5,

A

GUC ACOGGAAGUGI - I I In I I I I- aCOO U0ACCUUUoU

a helix 6G a C

spr -u aC-Qc-a

^ aA GA G G a

AaUUCUCCU~AA

3' domain

tobacco 16S rRNA

FIG. 3. Secondary structure of tobacco 16S rRNA, showing the major functional domains and sites of antibiotic resistance (Table 2; sr,streptomycin; spr, spectinomycin; nr, neamine/kanamycin). Sequence from the Ribosomal Database Project, courtesy of Robin Gutell.

the tertiary structure. Many of the same sites, which are all inhighly conserved regions of the 16S molecule, also interact withantibiotics that block protein synthesis at the level of the 30Ssubunit (424, 467, 537; also see below).The principal regions in which the secondary structures of

chloroplast 16S rRNAs deviate from the E. coli model are inthe 5' domain between nt 198 and 220 (numbering accordingto the E. coli sequence [462]), where chloroplast rRNAs havea shorter helix 10 than E. coli does, and between 455 and 477,where E. coli has a well-defined helix (the upper part of helix

0

nr

3' minor domain


17 of Brimacombe et al. [59]) that is lacking in cyanobacteriaand chloroplasts. Helix 6 is also shorter in chloroplast andcyanobacterial 16S rRNAs than inE. coli. Raue et al. (512)noted eight regions where secondary structure is conserved butprimary sequence is highly variable. These eight regions arealso sites of variation in secondary structure when the 16SrRNAs of chloroplasts and eubacteria on the one hand arecompared with small-subunit rRNAs of mitochondria andeukaryotic cytoplasmic ribosomes on the other.

23S rRNA

The secondary-structure model for the E. coli 23S rRNApublished by Gutell and Fox (233) consists of six domainscomprising a total of 95 helices (Fig. 4; for a numberedE. colisequence diagram in similar format to the tobacco sequenceshown in Fig. 4, see references 234 and 235). Domain V is theprincipal site of tRNA binding to the50S subunit (60, 426).The central loop of this domain is involved in the peptidyl-transferase center and is the site of mutations conferringresistance to erythromycin, lincomycin, and chloramphenicol(see below). Some tRNA interactions are found in domainsIIand IV. When bound to the P site, tRNA also interacts with the3' terminus of the 23S molecule (426). EF-G binds specificallyto position 1067 in the 23S molecule, a region identified with GTPhydrolysis (465). EF-Tu protects residues in the 2660 loop.Rau6 et al. (512) identified 18 variable regions in 23S RNAs

based on comparisons of eubacterial, organelle, archaebacte-rial, and eukaryotic large-subunit rRNAs, including the cya-nobacterium Anacystis nidulans and chloroplast 23S rRNAfrom Chlorella ellipsoidea, Marchantia polymorpha, tobacco andmaize. Of these 18 variable regions,5 are significantly differentin chloroplasts compared with E. coli, while in the remaining13 regions, chloroplast rRNAs resemble those of eubacteriabut may differ from those of archaebacteria, mitochondria, andeukaryotic cytoplasmic ribosomes. Somerville et al. (581) havepublished a secondary-structure map of the 23S rRNA fromthe brown alga Pylaiella littoralis which resembles the cya-nobacterial (Anacystis) molecule much more closely than itresembles those of land plants or green algae. Cladistic analysisof the 23S rRNA sequence produced a tree in which cyanobac-terial and plastid sequences were clearly delineated from allother eubacterial sequences and in which the chromophytealgae (as represented by Pylaiella littoralis) and Euglena gracilisformed a common branch.

In domain I, cyanobacterial and chloroplast 23S rRNAs lackhelix 8 of E. coli (nt 131 to 148, variable region V1) and havean insertion between helices 13 and 14 (E. coli nt 271 to 365,variable region V2) which can be folded into a helix (512). Indomain II, variable regions V4 and V7 (nt 636 to 655 and 1020to 1029, respectively) are highly conserved among eubacteriaand chloroplasts, while 3 nt (nt 931 to 933) in E. coli V6 arereplaced by a loop of 5 to 20 nt in chloroplasts. Region V8 (nt1164 to 1185) is conserved in E. coli, Anacystis nidulans, andmost chloroplast 23S rRNAs but is the site of a possible 243-ntintron in Chlorella ellipsoidea (726). Gutell and Fox (233) havesuggested that this insertion may actually be a part of therRNA rather than the only known instance of an introninserted in a variable rRNA region.Domain III comprises variable regions V9 to V12, of which

Vii (E. coli nt 1521 to 1542) is the most diverse in chloroplast23S rRNAs. Some (but not all) chloroplast rRNAs have lostpart of helix 54, and helix 55 in Chlorella ellipsoidea and Z.mays contains insertions compared with E. coli; however, inother chloroplast genomes, this helix is similar in size to that ofE. coli. Domain IV shows strong conservation among eubac-

teria and plastid rRNAs, as does domain V. The breakbetween the 23S rRNA and 4.5S rRNA of land plants occurs indomain VI.

5S rRNA

Their short length and relatively high degree of evolutionaryconservation have madeSS rRNA molecules frequent subjectsfor phylogenetic studies (see e.g., references 117, 270, 580, 663,664). They have also proved useful for computer modeling ofsecondary and tertiary structure, including chemical reactivityand accessibility of bases, and possible protein binding (67,524, 525, 693). A numbering scheme applicable to both pro-karyotic and eukaryoticSS rRNAs, proposed by Erdmann andWolters (157), defines five loops (a to e) and five helices (A toE). In a compilation of sequences in the Berlin RNA Data-bank, Specht et al. (583) included representations of thecommon secondary structure of eukaryotic and prokaryotic SSrRNAs, which are differentiated into five structural groupsprimarily on the basis of variability in one (D) of the fivehelices. PlastidSS rRNAs are grouped in this classification withthose of eubacteria and land plant mitochondria (mitochon-dria of other taxa lackSS rRNA). Plastid and cyanobacterial SSrRNAs are distinguished from those of most other eubacteriaand mitochondria by a single-base insertion in helix C and adeleted base in loop c (157). Of the 121 nt of the typical SSrRNA, 110 are identical in nearly all angiosperms and gymno-sperms, 73 are conserved in ferns and liverworts as well, and 29are identical in all plastids so far sequenced with a few singularexceptions. The colorless flagellate Astasia longa and the redalga P. umbilicalis are somewhat divergent compared withEuglena gracilis and P. purpurea, respectively; 4 nt are alteredin one or both of the two parasitic plants Conopholis americanaand Epifagus virginiana compared with all other angiosperms;and the sequence submitted to GenBank for cotton, Gos-sypium hirsutum (440), is missing 2 nt but is otherwise identicalto that of tobacco in all but two residues. Vogel et al. (671)reported that SS rRNA from spinach chloroplasts could beincorporated into biologically active 50S ribosomal subunitsassembled in vitro from Bacillus stearothermophilus proteinsand 23S rRNA.

Introns in rRNA Genes

A survey of 23S rRNA genes from 17 Chlamydomonasspecies representing most of the taxonomic groups defined onmorphological and biochemical grounds (159, 538) revealed atotal of 39 group I introns inserted at 12 different positions,some of which were unique to Chlamydomonas species (656-658). However, no correlation was found between introndistribution and a phylogeny for these 17 species based onprimary sequence of their 23S genes. Most of the introninsertion sites identified in this study are in highly conservedregions of the genome, which tend to be exposed in theassembled ribosome. This is also true of the single intron in the16S gene of C. moewusii, which lies within the 530 loop, a partof the translational fidelity domain. In contrast, internal tran-scribed spacers, which have also been identified in rRNA genesof bacteria and organelles, occur within regions of variableprimary sequence and secondary structure (224). When thesesequences are processed out of the pre-rRNA molecule, themature sequence is not religated, resulting in a fragmentedrRNA. Three internal transcribed spacers, found at equivalentpositions in the Chlamydomonas taxa studied by Turmel et al.(656-658), result in fragmentation of the 23S rRNA into fourmature rRNA species, ao, ,, -y, and &.The single group I intron in the 23S gene of C. reinhardtii

VOL. 58, 1994

710 HARRIS ET AL.

BAAU-A

GTPasecenter

V7

MICROBIOL. REV.

AAAUUaA cB cA u VI I

eUAAGAAG G tcc A AUUUC aI -*I I II I I I II I A

AAcUoUUUC%cccuu OGAAUBCAAA u

c cUUA Ue c

a UCOB AGGCGCCuG III,,|"UA ABCC a 0UC0CBOAA A _UBAAG

U-AU Au-C

AU A~~~~~~~~AA u~~~~~~~~~- caAU u au u 0-C0-C 6-CO-C A AA-U 0U0V9 AA -1 ViOA BA%CA B

GOCC 0ACQ%%.IA0AUaa4AusUC ZqGCCCCCUUBUUBaBUC CC0ABzG °scUAABBCPUA GUBUC BOA r BuuCUC AcA I1111 IIIIIII,I*I I CAa CCBA CUAAAU UG.LCACAG A ACC A

AGUCAUAAAf

'ac GUUUA1G1 AB-U A[q V12

C II 111111- AUUBG OCCUCCU' A

AAC U AC.UE

AACu ~~~domain IIIUB' A CC-B CA

_sC-B

uuC-Gc 3'fnfc

U A IUI-C A bAA_ _Ca

V6

domain I

V3

domain 11

tobacco 23S rRNA, 5'

e AAU-A

B-CC-B

AAC-BGBUUBUa

AA CAAA a c u

C=Ua-BV2

A-QU

oil 11|1 11,

0-2AAA'

._

FIG. 4. Secondary structure of tobacco 23S rRNA, showing major functional domains and sites of antibiotic resistance (Table 2). Variableregions are numbered according to the system of Raue et al. (512). Sequence from the Ribosomal Database Project, courtesy of Robin Gutell.


domain IV

V13

domain VI

tobacco 23S rRN

u

u

Ac 5' 4.5S UCA

cA UC CAUGG A G

_A G-C ACGGCGAGA 11111 A

U%GCCGA GcA UUAUA-UA C

U-A A

CC--G CAUUA -UC-G ~~~U

a-*U C

Aa-c uGUaA Ac

IA, 3 0C-GUaAa

C0 A

U GAA-U

U aGC-CU-A

CGU

U C

U * GC-aC-C

UC-G-C

a-CAAG A

Cuu V5 -CAICUA AG-CAC,1 LL 3UGG domainC UCACCTfCU U- UAGAGc-aU-AU-A

C-ac-aa-C GGAG u G GAGA A Au.G 0UU-A a u

tu V16 a

G-CUA Accuu U UCUCGGAC G-CCU -III I * I°I C G U GCA -UGACAG U-A Gu0-C G

aC A A C GOA AG

G - oc A CCAaa UC-aU

U-A A A CGc-a c c U-AC - a G C-O U U I-C

G *U GU cC-Ga G-1A- U G U: :A-

U-A0 G G-CUA -

U-A Uuca AA-U-c U: c ACUGC.C aUUC C

I I I IIIAU a ACG

CC

AA C aCU-A CG-C U-AAGUCUCA-aUu U-AA A A aAUA C G_.C GaO A AA GAC- GU

C GAUAC-G AU AACA

peptidyl GGCUGAUCUUCCCCACCU1I.1i1i1iI It C

transferase caUC GAAC A a Ac

UUC

3A aG c aa U.a

C=aCA- U

BC-U

Ua-CC-aAAGCCACCUGUGGCUG U

A 'AUtU G0GACCUUGUAUC-GC=G

.A C' CU'C UUG

(522) is mobile (142) and encodes a double-stranded nuclease(I-CreI) which has been purified and shown to have a 19- to24-bp recognition sequence in this gene (143, 638). Theenzyme makes a 4-bp staggered cut just downstream of theintron insertion site and will tolerate single- and even multiple-base-pair changes (143). This intron can undergo autocatalytic

splicing in vitro (637). The ac-20 nuclear gene mutant of C.reinhardtii, which was initially characterized as deficient inchloroplast ribosomes (see reference 247 and references there-in), has been found to accumulate unspliced precursor rRNAmolecules, as well as unspliced precursors of the chloroplast-encoded psbA gene (258, 259).

VOL. 58, 1994

IV

712 HARRIS ET AL.

The 23S rRNA gene of C. eugametos, a species now thoughtto be only distantly related to C. reinhardtii, contains six groupI introns and three internal transcribed spacers (657). Oneoptional 955-bp group I intron in the 23S gene of C. eugametosalso appears to be mobile and is transmitted to all progeny ofcrosses with the interfertile species C. moewusii, which lacksthis intron (347). The 402-bp group I intron in the 16S rRNAgene of C. moewusii likewise can be transmitted in crosses toisolates of the sibling species C. eugametos that lack this intron(140). Transmission of these introns is often accompanied bycoconversion of flanking DNA polymorphisms. The mobileintron in the 23S gene encodes a double-stranded DNAendonuclease activity (I-CeuI) which has a 19-bp recognitionsite centered around the insertion site. I-CeuI produces a

staggered cut 5 bp down from the insertion site (200, 406, 407).The 23S rRNA gene of C. humicola has a group I intron,ChLSU-1, inserted at a site in the peptidyltransferase loop andencoding a putative 218-amino-acid endonuclease (96). In-trons have been found at this site only in a few Chlamydomonasspecies (658).Turmel et al. (658) discuss the alternative possibilities for

transfer of group I introns from one site to another within a

genome. Intron-encoded endonucleases could effect such atransfer at the DNA level (139); alternatively, a reversal ofself-splicing followed by reverse transcription of the recom-bined RNA could occur, followed by integration into DNA byhomologous recombination. The latter mechanism requiresonly a short target site that can pair with the 5' intron sequencecalled the internal guide sequence (718) and would be consis-tent with the position of intron insertion sites in exposed rRNAregions in the ribosome in the Chlamydomonas species exam-ined by Turmel et al. (658).

The 16S-23S Spacer

The spacer regions between the 16S and 23S rRNA genes inchloroZlasts and cyanobacteria contain tRNAIleGAU andtRNA aUGcmas do the E. coli rmA, mD, and rmH operons(301, 436, 697, 735). In E. coli and cyanobacteria, the 16S-23Sspacer is short (<550 bp), but in land plants and charophytealgae it is 1 to 2 kb or more, largely because of the presence oftype II introns in the two spacer tRNA genes (110, 311, 320,394, 628). In all other algae so far examined, these spacertRNAs are uninterrupted (108, 216, 384, 405, 551, 725). In C.reinhardtii, an 1,100-bp region between the 3' end of the 16Sgene and tRNAIle contains short dispersed repeat elements indirect and inverted orientations, which are capable of pairingto generate extensive secondary structure in the precursorRNA (551). Similar repeat elements are found elsewhere inintergenic regions of the chloroplast genomes of C. reinhardtiiand the interfertile species C. smithii, and variations in theirnumbers are responsible for most of the restriction fragmentlength polymorphisms between the chloroplast genomes ofthese isolates (50, 250, 486). The absence of these repeats inchloroplast rRNA operons of other organisms, including C.eugametos and C. moewusii, and their variation in number inthe 16S-23S spacer region between C. reinhardtii and C. smithiisuggest they are not essential for processing of the rRNAprecursor.Although the 16S and 23S rRNA genes in the plastid

genome of the colorless euglenoid flagellate Astasia longa arehighly homologous to those of Euglena gracilis, the spacerbetween these genes appears to lack tRNAIle and tRNAAla(569). The chloroplast genome of Chlorella ellipsoidea was alsoreported to lack the spacer tRNAAla (724), but subsequentanalysis has shown that the rRNA operon in this alga has been

disrupted by an inversion of a 5-kb region with a breakpointbetween the two tRNAs, so that the 5S, 23S, and tRNAMagenes constitute a second operon on the opposite strand fromthe 16S and tRNAIle genes (723, 725).

tRNAs Flanking the rRNA Operons

Genes encoding tRNAs are also often found in regionsflanking the rRNA operons, but their presence and identity aremuch more variable than for the two tRNA genes in the16S-23S spacer. There is a tRNAVa" proximal to the 5' end ofthe 16S rRNA gene in all land plants examined (114). Thisgene, which precedes the promoter for the rRNA operon, isnot present in C. reinhardtii or C. moewusii, nor is it foundupstream of any of the E. coli rm operons or in any of thecyanobacterial sequences to date. In Euglena gracilis, theequivalent tRNAVal is in a gene cluster distant from the rRNAoperons (477), and a pseudo-tRNAIle is found 5' to the 16Sgene (479).

In land plants the 5S rRNA gene is typically followed by atRNAN9 gene in the same orientation and by a tRNA,sn geneon the opposite strand (118, 297, 298, 305, 557). In maize,primer extension experiments have shown that the tRNA'9gene, which is separated from the 5S gene by a 252-bp spacer,is cotranscribed with the rRNA operon (118). This operon andthe tRNA In gene, which is distal to tRNAArg by 253 bp on theopposite strand, are thought to share a common terminatorregion consisting of a palindromic sequence which can befolded into hairpin structures on both strands.

Antibiotic Resistance Mutations in theChloroplast rRNA Genes

Many antibiotics that inhibit bacterial protein synthesis bindspecifically to the 16S or 23S rRNA molecules (102, 424, 425),and mutants resistant to these antibiotics have been shown toresult from single-base-pair changes in evolutionarily con-served regions of the genes encoding these RNAs in bacteria,mitochondria, and chloroplasts (Table 2). Streptomycin resis-tance can result from changes at several nucleotides clusteredin three sites in the 16S chloroplast rRNA molecule of landplants and green algae (equivalent to E. coli residues 13, 523 to525, and 912 to 915). Although these three sites are widelyseparated in the primary sequence, they interact with the samesubset of ribosomal proteins and are thought to be in closeproximity in the assembled 30S subunit of E. coli (603).Spectinomycin resistance has been shown to result from mu-tations at the bases of the chloroplast 16S rRNA equivalent toE. coli residues 1191 to 1193 and at the base equivalent toresidue 1064, which pairs with 1192. Neamine and kanamycinresistance in C. reinhardtii can result from mutations at thechloroplast 16S rRNA nucleotides equivalent to E. coli resi-dues 1408 and 1409. In E. coli, binding of aminoglycosideantibiotics to this region has been demonstrated (424, 717),and site-directed mutagenesis of these and neighboring baseshas been used to obtain a number of mutants (116). Becausethe E. coli genome has seven rm operons, antibiotic resistancemutations must be selected by expression of cloned rRNAoperons on high-copy-number plasmids (570, 603). In contrast,an efficient copy correction mechanism involving the invertedrepeat ensures that newly occurring 16S mutations can spreadto both rRNA cistrons in the chloroplast genome (50).

Erythromycin resistance mutations in the large subunitrRNA are known in bacteria, in mitochondria of Saccharomy-ces cerevisiae and mammalian cells, and chloroplasts at the

MICROBIOL. REV.


TABLE 2. Antibiotic resistance mutations in chloroplast rRNA and ribosomal protein genes compared with analogous mutations in E. coliand mitochondria

Taxon' Nucleotides Reference(s)

16S rRNA mutations to streptomycin resistanceE. coli wild typeE. coli mutantE. coli mutantC. reinhardtii wild typeC. reinhardtii mutantE. coli wild typeE. coli mutantNicotiana plumbaginifolia wild typeN. plumbaginifolia mutantNicotiana tabacum wild typeN. tabacum mutantC. reinhardtii wild typeC. reinhardtii mutantC. eugametos wild typeC. eugametos mutantE. coli wild typeE. coli mutantE. coli mutantE. coli mutantE. coli mutantE. coli mutantE. coli mutantEuglena gracilis wild typeE. gracilis mutantMycobacterium tuberculosisM. tuberculosis resistant isolateNicotiana plumbaginifolia wild typeN. plumbaginifolia mutantN. tabacum wild typeN. tabacum mutantN. tabacum mutantC. reinhardtii wild typeC. reinhardtii mutantC. reinhardtii mutantC. reinhardtii mutant

16S rRNA mutations to spectinomycin resistanceN. tabacum wild typeN. tabacum mutantN. tabacum wild typeN. tabacum mutantN. tabacum mutantN. tabacum mutantC. reinhardtii wild typeC. reinhardtii mutantC reinhardtii mutantC. reinhardtii mutantE. coli wild typeE. coli mutantE. coli mutantE. coli mutantZea mays (naturally resistant)N. tabacum wild typeN. tabacum mutant

4 JUGAAGAGUUUGAUCAUG 214 . A. 214 . C. 215 AUGGAGAGUUUGAUCCUG 225. G. 22517 GCCAGCAGCCGCGGUAAU 534517 ......C........... 534465 GCCAGCAGCCGCGGUAAU 482465. U. 482464 GCCAGCAGCCGCGGUAAU 481464........U ......... 481468 GCCAGCAGCCGCGGUAAU 485468 ......C. .......... 485

GCCAGCAGCCGCGGUAAU...........C

905 UAAAACUCAAAUGA 918905 .. 918905. G. 918905. U 918905. C 918905 . ... 918905. ...G 918869 UGAAACUCAAAGGA 882869. U. 882858 UAAAACUCAAAGGA 871858. G. 871854 UGAAACUCAAAGGA 867854. U. 867853 UGAAACUCAAAGGA 866853. A. 866853. U. 866849 UGAAACUCAAAGGA 862849. U. 862849. C 862849 .G 862

10061006113311331133113311181118111811181186118611861186113213261326

16S rRNA mutations to neamine and kanamycin resistanceC. reinhardtii wild typeC. reinhardtii mutantC. reinhardtii mutant

133213321332

GCUGUCGUCAGC 1017...... .... 1017

GGAUGACGUCAAGU 1146..... .. 1146...........U.1146....... ..... 1146

GGAUGACGUCAAGU 1131.......1131.......1131

....... ..... 1131GGAUGACGUCAAGU 1199...........U.1199

...... .. ...G .1199...........A.1199

GGAUGAGGCCAAGU 1145GUUCCCGGGCCUUGUAC 1341....... ... .. 1341

CGCCCGUCACACCAUGGA 1349...............G.1349

......... ........1349

23S rRNA mutations to erythromycin and/or lincomycinresistance

C. reinhardtii wild typeC reinhardtii mutantC. reinhardtii mutantE. coli wild typeE. coli mutant

20072007200720502050

CUGGACAGAAAGACCC 2022....... ..... 2022

........ ...... 2022

CAAGACGGAAAGACCC 2065....... ..... 2065

34625125165158


6649349313725166420729643, 6446461841372511991996643134349337493343217428131131729643, 644646160184137251251251

646182646620620182137249, 251249, 251249, 2516426, 387, 57026, 38726, 387554646620

137251251

VOL. 58, 1994

714 HARRIS ET AL.

TABLE 2-Continued

Taxona Nucleotides Reference(s) 2

E. coli mutantE. coli mutantNicotiana plumbaginifolia wild typeN. plumbaginifolia mutantN. plumbaginifolia mutantSaccharomyces cerevisiae mitochondria wild typeS. cerevisiae mutantE. coli wild typeE. coli mutantC. reinhardtii wild typeC. reinhardtii mutantC. reinhardtii mutantC. moewusii wild typeC. moewusii wild typeS. cerevisiae mitochondria wild typeS. cerevisiae mutantS. cerevisiae mutant

2050............... 20652050............... 2065

CUGGACAGAAAGACCC........G................ ......

1943 GCAGACGGAAAGACCC 19581943 . . G....... 19582601 CAGUUCGGUCCCUAUC 26162601 ..........u ..... 26162559 CAGUUUGGUCCAUAUC 25742559 ..........U ..... 25742559 ..........G..... 2574

CAGUUUGGUCCAUAUC.......... .....

2775 CAGUAUGGUUCCUAUC 27902775......... G 27902775

2499249925092509267226722672

23S rRNA mutations to chloramphenicol resistanceE. coli wild typeE. coli mutantC. reinhardtii wild typeC. reinhardtii mutantS. cerevisiae mitochondria wild typeS. cerevisiae mutantS. cerevisiae mutant

S12 mutations to streptomycin resistance and dependenceE. coli wild typeE. coli mutant (sr)E. coli mutant (sr)E. coli mutant (sr)E. coli mutant (sr)C. reinhardtii wild typeC. reinhardtii mutant (sr)E. coli wild typeE. coli mutant (sd)E. coli mutant (sr)E. coli mutant (sd)E. coli mutant (sd)C. reinhardtii wild typeC. reinhardtii mutant (sd)N. plumbaginifolia wild typeN. plumbaginifolia mutant (sr)N. tabacum wild tpeN. tabacum mutant (sr)a sr, streptomycin resistance; sd, streptomycin dependence.

...............

CUCGAUGUCGG 2509......... 2509

CUCGAUGUCGG 2519..... .. 2519

CUCGAUGUCGA 2682.... ...2682..... ... 2682

2790

38 TTTPKKPNSA 4738 .... N. 4738 . Q..Q. 4738 .... R. 4738 .... T. 4738 TVTPKKPNSA 4738 .... T. 4783 GGRVKDLPGV 9283 .S.. 9283 .... R. 9283. L 9283. D. 9283 GGRVKDLPGV 9283. L 9283 GGRVKDLPGV 9283 .... R. 9283 GGRVKDLPGV 9283 .S 92

positions equivalent to E. coli nt 2057 to 2058 (the yeast rib3

locus) and 2611 (yeast rib2) (Table 2). Some of the erythro-mycin-resistant mutants of C. reinhardtii are cross-resistant tolincomycin. A lincomycin-resistant mutant of Nicotiana plum-baginifolia has also been identified at the base equivalent to E.coli nt 2032. Chloramphenicol resistance in C. reinhardtiiresults from a nucleotide substitution at a position equivalentto E. coli nt 2504 (208). Chloramphenicol resistance mutationsat this site are also known in mitochondria of yeast (the rbllocus) and mammals. All three regions of conserved sequence

together form a loop known to be involved in peptidyltrans-ferase activity in E. coli (464).

Chloroplast antibiotic resistance mutations have been usedas markers in generation of transgenic tobacco plants by usingsomatic cell fusions (427) and in chloroplast transformation ofChlamydomonas species (48, 49, 453) and of tobacco (390, 619)by using biolistic techniques.

RIBOSOMAL PROTEINS

Number and NomenclatureRecent reviews provide an overview of chloroplast ribo-

somal proteins and the genes that encode them (36, 377, 379,606, 608, 609, 615). For a concise summary of structure andfunction of individual ribosomal proteins, with emphasis on theE. coli ribosome, see the review by Liljas (358). Lindahl andZengel (360) provide a review of bacterial genes for ribosomalproteins. Chloroplast ribosomal proteins were initially identi-fied from various plants and algae by one-dimensional andtwo-dimensional gel electrophoresis and were numbered ac-

cording to their migration on these gels, a function of chargeand/or molecular mass, depending on the gel system. Naturalvariations in the physical properties of ribosomal proteinsthemselves, together with differing electrophoretic conditionsused for their separation, have meant that no two numbering

570132999999See 100See 10065667346251251199199See 100See 100100

65158, 425346208See 158See 158See 158

707188190188190364364707285188662285364364276276561191

MICROBIOL. REV.


schemes are the same. Even the well-characterized ribosomalproteins of E. coli are not numbered according to theirmigration on two-dimensional sodium dodecyl sulfate-poly-acrylamide gels. Since most of the chloroplast ribosomalproteins identified on gels have not yet been correlated withsequenced genes, organism-specific terminologies are some-times used, e.g., the numbering system of Mache et al. (378)and Dorne et al. (119, 120) for spinach, the system of Capeland Bourque (73) for tobacco, and that of Schmidt et al. (547)for C. reinhardtii. When used here, such designations will begiven in quotation marks as "L-13," etc. However, in cases inwhich equivalence has been established, designations of thechloroplast ribosomal proteins and the genes encoding theseproteins have been changed to indicate the E. coli ribosomalproteins to which they correspond. For example, seven of theribosomal proteins from chloroplast ribosomes of spinach havebeen purified by Schmidt et al. (540) and shown by N-terminalsequencing to be equivalent to seven E. coli proteins (S12, S16,S19, L20, L32, L33, and L36), whose homologs are encoded bychloroplast DNA. Those chloroplast and nuclear genes encod-ing chloroplast ribosomal proteins corresponding to those of E.coli are designated rps- or rpl- followed by numbers equivalentto the similar bacterial ribosomal protein designation (S1 toS21 for small-subunit ribosomal proteins and Li to L36 forlarge-subunit ribosomal proteins [241, 606]). Thus, rps4 en-codes protein S4 and corresponds to E. coli rpsD, rpl2 encodesprotein L2 (E. coli rplB), etc.

Previous estimates of the number of chloroplast ribosomalproteins in the small and large subunits have been in the rangeof 22 to 31 and 32 to 36, respectively (73, 119, 156, 495, 547),i.e., at least as many as in E. coli, in which 21 and 33 proteinshave been identified in the small and large subunits, respec-tively. Part of the variability in these estimates is undoubtedlythe result of differing isolation and electrophoretic conditions.In some circumstances these factors may cause certain pro-teins, particularly those of higher molecular weight, to beexcluded from gels (see references 510 and 547 for discussion).

Organization of Chloroplast Ribosomal Protein Genes

The first suggestion that chloroplast genomes might encodechloroplast ribosomal proteins came from labeling experi-ments carried out in the presence of inhibitors specific foreither chloroplast or cytoplasmic protein synthesis. Thesestudies led to the then remarkable conclusion that in landplants, Euglena gracdiis, and C. reinhardtii about one-third of thechloroplast ribosomal proteins were themselves synthesized- onchloroplast ribosomes, with the remainder being made on cyto-plasmic ribosomes and imported (121, 156, 181, 239, 495, 547).With a few notable exceptions, the same subset of ribosomal

proteins is encoded in the chloroplast genome of each landplant examined, while marked variations occur in certain algalgroups (see below). The genes encoding many of these pro-teins are arranged in clusters that are clearly remnants of theribosomal protein operons of eubacteria (Fig. 5) (see refer-ences 36, 377, 606, and 608 for additional discussion). ThePorphyra plastid genome has the most complete version ofthese operons found to date (Fig. 5; Table 3) (514, 517). Mostof the same genes are also present in the cyanelle genome, butthe operon has been broken into three pieces (598). In landplant chloroplasts the largest cluster contains the genes forribosomal proteins L23, L2, S19, L22, S3, L16, L14, S8, L36,and S11 and the RNA polymerase gene rpoA in the same orderthat they appear in the E. coli S10, spc and a operons, whichare part of the str cluster (Fig. 5; see Table 3 for references). Asimilar organization of genes encoding ribosomal proteins is

seen in the Marchantia mitochondrial genome (630). The rps4and rpsl4 genes are also found in chloroplast genomes of landplants but are relocated outside the ribosomal protein cluster.In some legumes, rp122 has been removed from this cluster andrelocated to the nucleus (193), and in a number of dicots thechloroplast rp123 gene is disrupted and probably nonfunctional(see below). The rp122 gene is also missing from this operon inChlamydomonas species (43, 277). In Euglena gracilis (242) andin both C. reinhardtii and C. moewusii (43, 277), the ribosomalprotein gene cluster also contains rplS, which does not appearto be present in the chloroplast genomes of land plants.However, the Euglena operon lacks rpsll, which is now in aseparate operon with rps4 (242, 597). The Chlamydomonasoperons lack rps3, but open reading frames with homology torps3 are found elsewhere in the chloroplast genome (seebelow). In C. moewusii, the large ribosomal protein cluster hasbeen disrupted by a rearrangement such that rp123, rpl2, andrpsl9 are separated from rplJ6, rpll4, rplS, and rps8 by about 42kb (43). The genes encoding S17, L24, and L15, which are partof these operons in bacteria, have been identified in thenuclear genomes of certain land plants (153, 196, 640). Theremaining genes of these E. coli ribosomal protein operons(encoding proteins L17 and L30) have not been identified withplastid equivalents thus far.

In E. coli, the genes encoding S12, S7, and the elongationfactors EF-G and EF-Tu constitute a fourth operon in the strcluster (Fig. 5). This operon has undergone several alterationsin the course of plant evolution. It persists intact in cyanobac-teria (422, 641), but the fiusA gene encoding EF-G is absentfrom all chloroplast genomes analyzed so far and has presum-ably been relocated to the nucleus. In Cyanophora paradoxa,the cyanelle str operon includes rpsl2, rps7, tufA, and rpslO,which are processed from a primary transcript into two dicis-tronic mRNAs (68, 69, 368). The rpslO gene is also down-stream from tufA in Porphyra and Cryptomonas species (517).In the Euglena chloroplast, the rpsl2 and rps7 genes constituteone operon and the tufA gene remains adjacent but is sepa-rately transcribed (430). In land plants, where tufA is a nucleargene (15), the rpsl2 gene has been split, with the second andthird exons remaining proximal to rps7 and the first exonencoded separately downstream from rpl20 (183, 187, 649).Lew and Manhart (352) have recently reported that the rpsl2gene is also split in a green alga, Spirogyra maxima. This alga isbelieved to represent a relatively early stage in the charophytelineage leading to land plants. The rpsl2 mRNA is assembledby trans-splicing (264, 313, 737). In three species of theangiosperm genus Anemone, the second rpsl2 intron has beenlost secondarily, in conjunction with expansion of the invertedrepeat and several inversions within the chloroplast genome(269).

In two Chlamydomonas species, the rpsl2 operon has beenfurther disrupted. In C. reinhardtii, the uninterrupted rpsl2gene (364) is separated from tufA by about 40 kb and iscotranscribed with the psbJ and atpI genes encoding photosyn-thetic proteins (253, 572). The entire rps7 gene is located in theother single-copy region about 50 kb away, 5' to and cotrans-cribed with the atpE gene (253). In C. moewusii, rpsl2, rps7,and tufA are also widely separated; both rps7 and rpsl2 havebeen completely sequenced and are uninterrupted (655).Most of the remaining chloroplast-encoded genes for ribo-

somal proteins are transcribed either separately or in operonsthat also contain genes for photosynthetic proteins (606, 608).The rp133 and rpsl8 genes are cotranscribed in land plants,whereas the corresponding genes in E. coli are each part of a

different operon. The rpl21 gene, which is monocistronic in theMarchantia chloroplast genome (312), has not been found in

VOL. 58, 1994

716 HARRIS ET AL.

812DL EJuf81 IEI812j[E E.Ji]

812' 1!

S10

810 D18 123 810L225183 IL16 129

810L3 L2 81L22 83 I L16 8A

81018L4L23 L2 EjJiJ1 83 16 12A

Ri~JEE] 819 1 83 1L16

123 12 [ [GITiSi3| 16123T~J 19 i~jr~I3I j1

apC alpha1~~~~

15ER|814 88 16118 85 130IE &K1S lIlI 136 I 8131111 || S4 IiFPOAII 117 I

EjE7 rEli 88 16 116 85

114 88ELE16 188 EREEJ~~ElbElJ_rpo

rpo

Euglons E1 E _JE

C. relnhardtll

MarchantlaEE1

Epifagus _136 |

Nicotlana ESFIG. 5. Conservation of ribosomal protein gene clusters in chloroplast genomes, showing retention of some (but not all) genes of the closely

adjacent str, S10, spc, and a operons of E. coli (12), in Cyanophora paradoxa (598), P. purpurea (514, 517), Euglena gracilis (243), C reinhardtii (43,277), Marchantia polymorpha (471), Epifagus virginiana (712, 714), and Nicotiana tabacum (560). Shaded boxes indicate genes that have been lostfrom the corresponding operon but have been identified elsewhere in a given plastid genome. For example, rps7 and tuf4 are present in the Creinhardtii chloroplast genome but have become separated from rpsl2. In Cyanophora paradoxa and P. purpurea, the operon begins with the rpl3gene (A) and ends with the rpsl2, rps7, tufA, and rpslO genes (517, 598). The rpsl2 gene in land plants is split, and the 3' portion of the gene remainsproximal to rps7.

the Euglena, rice, or tobacco chloroplast genomes (243, 265,560) and has been identified as a nuclear gene in spinach (408,578). Conversely, rpsl6 is a chloroplast gene in all angiospermsso far examined and in Euglena, Cyanophora, and Porphyraspecies (Table 3) but is absent from the Marchantia and Pinusthunbergii chloroplast genomes (614, 654).

In E. coli and Bacillus subtilis, as well as in the cyanobacte-rium Synechocystis sp., the genes encoding Li, L10, Lll, andL12 are clustered (539, 564). The rpll, rplll, and rp112 genesalso form a cluster in the cyanelle genome, but rpllO isapparently missing (32). The rpoB and rpoC genes, which arepart of the same cluster in E. coli, were found elsewhere in theSynechocystis genome and in the cyanelle genome. None ofthese four ribosomal protein genes has been found in any landplant chloroplast genome, but rpl12 is now known to be achloroplast gene in Euglena gracilis, located some distancefrom the rpoB and rpoC genes (243).

Correspondence of Chloroplast Ribosomal Proteins toBacterial Ribosomal Proteins

Of the 54 ribosomal proteins that constitute the E. coliribosome, the chloroplast equivalents of 44 have been identi-fied by sequencing of nuclear or chloroplast genes from one ormore organisms (Table 3). Derived amino acid sequenceidentities for these proteins with their equivalents in E. coli are

mostly in the range of 35 to 55%, with S12 showing consider-ably greater conservation (Table 3). In addition, at least three

distinct genes for chloroplast ribosomal proteins that show noobvious sequence similarity with any bacterial ribosomal pro-tein have been found in the nuclear genomes of pea andspinach (192, 290, 741).

In general, the chloroplast-encoded ribosomal proteins showgreater immunological cross-reactivity with bacterial ribosomalproteins than those encoded in nuclear genes (510). Thechloroplast-encoded proteins also show somewhat greatersequence identity to their counterparts fromE. coli than do thenucleus-encoded ones (Table 3). Subramanian et al. (608)made the interesting point that of 15 ribosomal proteins thatcan be individually eliminated by mutation in E. coli withouttotal loss of viability (103, 104), the equivalent of only one, L33,is chloroplast encoded in land plants. They suggest thatlocation of particular chloroplast ribosomal protein genes tothe nuclear or chloroplast genome may be related to theessential roles of these proteins in ribosome assembly or function.

In the following section we discuss each ribosomal protein inturn, briefly describing what is known about its function andstructure in bacteria and indicating the chloroplast equivalentsthat have been identified. Table 3 provides a summary ofreferences for sequence information to complement this text.

Proteins of the Small Subunit

Protein SI is essential for mRNA binding in E. coli and mayplay an important role in initiation of translation of mRNAsthat lack a Shine-Dalgarno sequence (604, 666). The gene

strE. coil operons

812 LI fE tf

Cyanophora

Porphyra

Euglena

C. relnhardtll

Marchantla

Epifagus

Nicotlana 8 i

E coil operons

Cyanophora

Porphyra

MICROBIOL. REV.


TABLE 3. Chloroplast and cyanobacterial ribosomal proteins that have been identified by gene or protein sequence

E. N.Proteina Taxon GenBank accession no(s). Locationa colib tabacumb Reference(s)

%S %I %S %I

Si Porphvra purpurea C 49 26 59c 38c 517-Spra -a oeraeaSpinacia oleracea X66135, M82923 N 48 22 100 100 178

Astasia longaConopholis americanaCyanophora paradoxaEpifagus vitginianaEuglena gracilisMarchantia polymorphaNicotiana tabacumOryza sativaPisum sativumPorphyra purpureaSpinacia oleraceaSpirulina platensisTriticum aestivumZea mays

Chlamydomonas reinhardtii 5'Chlamydomonas reinhardtii 3'Cyanophora paradoxaEpifagus virginianaEuglena gracilisGracilaria tenuistipitataMarchantia polymorphaNicotiana tabacumOryza sativaPorphyra purpureaSpinacia oleraceaZea mays

Chlamydomonas reinhardtiiChlorella ellipsoideaCryptomonas 4)Cyanophora paradoxaEpifagus virginianaEuglena gracilisMarchantia polymorphaNicotiana tabacumOryza sativaPorphyra purpureaSpinacia oleraceaZea mays

Cyanophora paradoxaPorphyra purpurea

Porphyra purpurea

Anacystis nidulansAstasia longaChlamydomonas moewusiiChlamydomonas reinhardtiiCryptomonas 4)Cuscuta reflexaCyanophora paradoxaEpifagus virginianaEuglena gracilisGlycine maxMarchantia polymorphaNicotiana tabacumOryza sativaPorphyra purpureaSpirodela oligorhizaSpirogyra maximaSpirulina platensis

X16004, X75651X64567 (partial)

X61798, M81884X70810, Z11874X04465Z00044X15901X05917, X03912

X05916X53651M35396X17318, X52270

CCCCCCCCCCC

CC

X66250X66250M30487M81884X70810, Z11874, M37463M32638X04465Z00044X15901

X13336Y00340, M31336

D10997X51511

M81884X70810, Z11874, M22010X04465Z00044X15901

M16878X01608

M30487

52 31 56 33 566634

66 48 65 49 59858 38 92 85 435,712,71457 35 60 40 88, 24363 45 83 73 65961 41 100 100 560, 56158 37 87 79 26561 42 94 87 97, 27866 47 70 50 51761 40 95 92 27884 71 60 42 53659 39 86 78 26759 39 87 79 282, 587, 588

C 44 25 49 25 172, 366C 57 36 56 34 172, 366C 72 51 67 47 423C 60 35 84 75 712,714C 69 51 58 35 88, 243C 64 46 65 43 294C 67 43 76 62 186C 64 39 100 100 560, 561C 66 40 83 70 265C 68 51 69 46 517C 65 42 95 89 742C 65 40 84 72 417

C 59 38 64C 65 44 66C 64 44 72C 63 41 74C 56 35 85C 56 35 70C 61 38 83C 58 36 100C 61 38 89C 62 38 72C 58 38 95C 59 38 88

C 64 42C 65 39

C 57 28

X17442X14385, X75652

X53977 (partial; see text)X52912X72584X52497M81884X70810, X06254, X00480X07675, X05013X04465Z00044, M19073X15901

X04508 (partial)L07932X15646

48 277, 51152 73458 12759 3276 712, 71451 24376 659100 560, 56180 26556 51792 22, 74279 610

423517

517

69 54 72 52 422C 48 26 50 29 565C 60 38 64 42 655C 56 37 63 44 509, 519C 69 49 70 55 122C 62 43 100 98 237C 67 49 71 53 326C 62 43 95 91 712, 714C 62 40 62 41 243, 430C 63 43 99 97 672C 66 43 88 78 659C 62 43 100 100 560,561C 60 43 92 85 265C 67 48 72 58 517C 498C 66 48 82 71 352

69 55 72 54 72


S2

S3

S4

S5

S6

S7

VOL. 58, 1994

718 HARRIS ET AL.

TABLE 3-Continued

E. N.Protein' Taxon GenBank accession no(s). Location" colib tabacumb Reference(s)

%S %I %S %I

Zea mays M17841 C 60 43 92 85 204

Astasia longaChlamydomonas reinhardtiiCyanophora paradoxaCyanophora paradoxaEpifagus virginianaEuglena gracilisMarchantia polymorphaNicotiana tabacumOenothera ammophilaOryza sativaPorphyra purpureaSpinacia oleraceaZea mays

C 60 34 64 38 566C 65 44 69 53 277

X16004, X75651

X16548M30487M81884X70810, Z11874X04465Z00044M60180 (partial)X15901

C 69 46C 68 46C 62 38C 62 48C 67 46C 65 42cC 62 41C 72 51C 53 37C 62 41

X13336X06734

67 47 6967 47 42389 84 712, 71464 44 88, 24378 59 186100 100 560, 561

71586 76 26568 47 51789 79 74286 79 400

Cryptomonas 4DCyanophora paradoxaEuglena gracilisPorphyra purpurea

Cryptomonas 4DCyanophora paradoxaPorphyra purpurea

Cyanophora paradoxaEpifagus virginianaEuglena gracilisMarchantia polymorphaNicotiana tabacumOryza sativaPisum sativumPorphyra purpureaSpinacia oleraceaZea mays

Anacystis nidulansChlamydomonas reinhardtiiCryptomonas 4DCuscuta refleaCyanophora paradoxaEpifagus virginianaEuglena gracilisGlycine maxMarchantia polymorphaNicotiana plumbaginifoliaNicotiana tabacumOryza sativaPinus contortaPorphyra purpureaSpinacia oleraceaSpirodela oligorhizaSpirogyra maximaSpinulina platensisTriticum aestivumZea mays


Astasia longaChlorella ellipsoideaChlorella-like algaCyanophora paradoxaEpifagus virginianaEuglena gracilis

X52912 (partial)

X70810

X52912X52143, M35206

cccc

62 4262 4164 44

C 71 52C 71 50C 72 49

M81884X70810, Z11874, M22010X04465Z00044X15901X15645, X05029

X03496M35831

X17442M29284X52912X72584 (partial)X52497M81884X70810, X00480, X06254X07675, X05013X04465, X03661, X03698L12250, L12366X03481, Z00044X15901L28807 (partial)

(partial)X04508 (partial)L07931, L07932X15646X54484 (partial)X60548, M17841, M17842

C 72 53C 71 54C 58 38C 72 51C 71 55C 72 52C 72 54C 72 55C 72 54C 72 52

122598243517

12268, 451, 452517

72 55 59890 83 712, 71469 44 243, 47789 79 186, 659100 100 560, 56187 74 26593 84 504, 50674 55 51799 90 57188 73 401

82 74 89 81 422C 76 68 84 78 364CCCCCCCCCCCCCCC

CC

82 73 90 84 122238

83 77 88 84 32677 67 92 90 712, 71482 69 85 73 243, 43078 70 98 98 67281 71 94 92 187, 65979 71 100 100 27679 71 - d d 183, 560, 56179 67 94 89 265

8980 73 88 80 517

540498

81 73 90 87 35282 74 88 81 72

21879 67 93 89 204, 687

C 71 54C 73 52

X16004, X75651D10997M74441, M81884

X61798X70810, Z11874, X15240

598517

C 62 46 68 55 566C 65 50 67 52 734C 64 48 65 52 6C 65 46 71 54 598C 56 39 94 90 435, 712, 714C 61 42 61 46 88, 243

Continued on followingpage

S8

S9

S10

Sil

S12

S13

S14

MICROBIOL. REV.


TABLE 3-Continued

E. N.Proteina Taxon GenBank accession no(s). Locationa col tabacumb Reference(s)

%S %I %S %I

Marchantia polymorphaNicotiana tabacumOryza sativaPisum sativumPorphyra purpureaSpinacia oleraceaZea mays

Marchantia polymorphaNicotiana tabacumOryza sativaSecale cerealeZea mays

Cyanidium caldariumHordeum vulgareNicotiana tabacumOryza sativaPorphyra purpureaSinapis albaSolanum tuberosumZea mays

Arabidopsis thalianaPisum sativumPorphyra purpurea

X04465Z00044X15901, X13208X05394

X04131Y00359, M16559

X04465Z00044X15901X14811X52614

X62578X52765Z00044, X03415X15901

X13609Z11741 (partial)X60823

J05215, Z11151M31025

C 58 44 82 75 659C 56 41 100 100 560, 561C 56 40 92 87 95, 265C 57 38 85 79 344C 56 40 70 56 517C 54 40 90 90 307C 56 40 92 87 523,586

C 53 36 72 58 312C 61 36 100 100 560, 561C 58 36 82 71 265C 59 38 81 69 501C 60 41 86 77 170

C 59 39 63 41 385C 62 38 90 86 556C 60 37 100 100 560, 561C 66 39 87 82 265C 58 42 71 51 517C 62 39 87 81 450C 138C 63 37 90 84 293

N 61 34 72e 55e 196, 640N 54 29 100 100 195, 196C 69 49 54e 39e 517

Chlamydomonas reinhardtiiCyanophora paradoxaEpifagus viginianaEuglena gracilisMarchantia polymorphaNicotiana tabacumOryza sativaPorphyra purpureaZea mays

Astasia longaChlamydomonas reinhardtiiCyanophora paradoxaEpifagus virginianaEuglena gracilisGlycine maxMarchantia polymorphaNicotiana debneyiNicotiana tabacumOryza sativaPetunia hybridaPisum sativumPorphyra purpureaSinapis albaSpinacia oleraceaZea mays


X17498M81884X70810, Z11874X04465Z00044X15901

X56673

C 53 34 63 41 348C 69 47 68 54 163C 58 34 86 81 712, 714C 61 37 70 48 134C 58 38 88 74 186C 56 37 100 100 560, 561C 55 34 79 72 265C 69 48 72 51 517C 55 34 75 70 686

X75653

X17498M81884X70810, Z11874, M37463X06429X04465

Z00044, V00163X15901M35955, M37322 (partial)X59015

X17331 (partial)X13336, X00797Y00141

C 63 44 66C 78 67 81C 83 70 85C 65 47 84C 71 57 72C 75 54 97C 77 62 88C 75 56 100C 75 56 100C 65 45 84CC 73 55 88C 78 62 76CC 76 57 97C 65 45 82

C 53 34C 51 33

43 21162 27768 16383 712, 71454 88, 24392 58480 186100 745100 560, 561, 61270 265

384 44860 517

45492 635, 74570 415

32517

S2lf

Ll Cyanophora paradoxaPorphyra purpureaSpinacia oleraceaSynechocystis strain PCC

6803

C 65 45 32C 62 41 517N 64 43 300

X73005 61 46 539

C 66 51 71 53 211


S15

S16

S17

S18

S19

S20

L2 Astasia longa X75653

VOL. 58, 1994

720 HARRIS ET AL.

TABLE 3-Continued


%S %I %S %I

Chlamydomonas reinhardtiiCyanophora paradoxaEpifagus virginianaEuglena gracilisGlycine maxMarchantia polymorphaNicotiana debneyiNicotiana tabacumOryza sativaPetunia hybridaPisum sativumPorphyra purpureaSinapis albaSpinacia oleraceaTriticum aestivumZea mays

X17498M81884X70810, Z11874, M37463X06429 (partial)X04465X00798Z00044X15901M35944, M37322 (partial)X59015

X65615X00797(partial)X53066, X12851, X62070

CCCCCCCCCCCCCCCC

70 53 75 60 27768 52 76 61 16364 47 96 93 712, 71468 52 72 55 88, 243

58464 49 81 73 18660 43 91 88 74566 48 100 100 560, 56164 49 93 90 265

368 50 94 93 44867 50 73 59 51766 48 97 97 45563 44 90 85 745

4665 49 93 90 299


Porphyra purpurea

Astasia longaChlamydomonas reinhardtiiCyanophora paradoxaCyanophora paradoxaEuglena gracilisPorphyra purpurea


X17498

X16004, X14384, X75651

X16548M30487X70810, Z11874, X17051

X16548, M30487

L7 (see L12)

L8 (see L10)

L9 Arabidopsis thalianaPisum sativumPorphyra purpureaSynechococcus sp.Synechocystis strain PCC

6803

Z11509, Z11129X14019

X63765D10716

N 53 32 82e 69e 640N 54 34 100 100 192C 61 30 55e 33e 517

56 36 58e 34e 43957 34 62~ 38e 388

Synechocystis strain PCC6803

Arabidopsis thalianaCyanophora paradoxaPorphyrapurpureaSpinacia oleraceaSynechocystis strain PCC

6803

Arabidopsis thalianaArabidopsis thalianaArabidopsis thalianaCyanophora paradoxaEuglena gracilisNicotiana sylvestrisNicotiana tabacumNicotiana tabacumPorphyra purpureaSecale cerealeSecale cerealeSpinacia oleracea

N 63 51 93C 88C 543C 71 55 75C 63c 32C 70 54 80c 65c 517N 63 52 100 100 579

75 61 82C 69C 539X56615X73005

X68046 (a)X68046 (b)X68046 (c)

X70810S93166X62368X62339

X68325X68340J02849

N 69 46 86 75 543, 689N 64 40 71 59 543, 689N 69 46 86 75 543, 689C 68 47 63 39 32C 66 47 63 44 243N 69 48 99 99 354N 70 48 99 99 155N 70 49 100 100 155C 74 58 70 49 517N 70 45 82 70 544N 69 44 80 68 544N 75 53 89 78 201


L3

L4

L5

C 62 45C 63 45

L6

C 60 38

161517

517

5662776942388, 243517

69, 423517

C 66 44C 76 51C 76 51C 76 52C 69 44C 74 54

C 63 38C 58 41

Lll

X53178

L12

53 26 539, 564

MICROBIOL. REV.

L10


TABLE 3-Continued

E. N.

Proteina Taxon GenBank accession no(s). Locationa coli' tabacumb Reference(s)%S %I %S %I

Synechocystis strain PCC X53178, X67516 78 62 70 45 539, 5646803

Porphyra purpureaSpinacia oleracea

Chlamydomonas reinhardtiiCyanophora paradoxaEuglena gracilisMarchantia polymorphaNicotiana tabacumOenothera ammophilaOryza sativaPorphyra purpureaSpinacia oleraceaVigna unguiculataZea mays

Arabidopsis thalianaPisum sativum

Chlamydomonas reinhardtiiCyanophora paradoxaEpifagus virginianaEuglena gracilisGracilaria tenuistipitataMarchantia polymorphaNicotiana tabacumOenothera ammophila0ryza sativaPorphyra purpureaSpinacia oleraceaSpirodela oligorhizaVigna unguiculataZea mays

C 66 51 69C 57C 517J04461 N 71 54 100 100 490

X14062M30487X70810, Z11874X04465Z00044M60179, M60180 (partial)X15901

X13336M80799 (partial)X06734

Z11507, Z11508Z11510

M13931M30487M81884X70810, Z11874M32638X04465Z00044M60179 (partial)X15901

C 77 57 88 71 372C 80 58 89 69 423C 76 60 85 63 88, 243C 83 58 94 80 186C 80 55 100 100 560, 561C 715C 80 54 93 85 265C 80 60 85 67 517C 82 58 94 88 742C 8C 80 53 92 81 400

N 61 41 76e 68e 640N 63 44 100 100 640

CCCCCCCCCCCCCC

X13336X03834M80799 (partial)Y00375, X06734 (partial)

79 57 82 69 37376 54 88 70 42376 50 90 81 712, 71473 53 77 65 88, 24372 60 77 65 29475 56 90 79 18678 56 100 100 560, 561

71576 54 90 86 26572 55 81 68 51776 53 96 90 74275 54 90 85 497

8400, 418


Cyanophora paradoxaPorphyra purpureaSynechocystis strain PCC

6803

M30487 C 62 49C 65 45

423517

C 69 46 598C 69 47 517

X72627 72 56 542

Astasia longaChlamydomonas reinhardtiiCyanophora paradoxaEpifagus virginianaEuglena gracilisGlycine maxMarchantia polymorphaNicotiana tabacumOryza sativaPorphyra purpureaZea mays

Cyanidium caldariumCyanophora paradoxaMarchantia polymorphaPorphyra purpureaSpinacia oleracea

Astasia longaCyanophora paradoxa

X75653X62566X17063M81884X70810, Z11874, Y00143X07676 (partial)X04465Z00044X15901

X60548

X04465

M57413, M64682

X75653M30487, X17498

C 56 33 55 27 211C 66 46 68 44 736C 72 53 71 52 69C 60 41 81 77 712,714C 55 29 54 31 243, 396C 673C 64 45 76 57 186C 63 41 100 100 560, 561C 59 41 78 67 265C 69 47 64 46 517C 58 41 81 71 687

C 54 26 54C 32C 385C 58 31 57C 30C 598C 54 29 55C 30c 312C 62 33 52C 32C 517N 60 32 100 100 340, 578

C 53 31 60 34 211C 62 44 70 47 163, 423


L13

L14

L15

L16

L17f

L18

L19

L20

L21

L22

VOL. 58, 1994

722 HARRIS ET AL.

TABLE 3-Continued


%S %I %S %I

Euglena gracilisGracilaria tenuistipitataMarchantia polymorphaNicotiana tabacumOryza sativaPelargonium zonalePisum sativumPorphyra purpureaZea mays

Arabidopsis thalianaAstasia longaChlamydomonas reinhardtiiEuglena gracilisMarchantia polymorphaNicotiana tabacumOryza sativaPorphyra purpureaSinapis albaSpinacia oleraceaTriticum aestivumZea mays

Nicotiana tabacumPisum sativumSpinacia oleraceaPorphyra purpurea

X70810, Z11874, M37463M32638X04465Z00044X15901M60953M60951, M60952

Y00329

X66414X75653

X70810, Z11874, M37463X04465Z00044X15901

X65615X07462 (pseudogene?)X12850X07864

X14020M58522

C 59 38 52 42 88, 243C 59 40 64 42 295C 59 41 68 54 186C 54 36 100 100 560, 561C 53 32 70 54 265C 50 35 78 67 193N 58 43 72 53 193C 66 49 66 48 517C 47 28 66 52 416

C 50 25 97 95 374C 38 18 42 21 211C 53 31 55 39 277C 42 17 53 32 88, 243C 46 25 77 55 186C 48 23 100 100 560, 561C 51 26 94 84 265C 46 28 63 36 517C 50 25 98 96 455C 635C 51 26 94 83 46C 51 26 93 84 419

N 64 38 100 100 153N 56 34 86 78 192N 58 34 76 61 75C 51 33 61 49 517

Calyptrosphaera sphaeroideaChlamydomonas reinhardtiiChrysochromulina aliferaChrysochromulina hirtaCyanidium caldariumNicotiana tabacumPleurochrysis carteraePleurochrysis haptonemoferaPorphyra purpureaPorphyridium cruentum

D26097 (partial)N-terminal amino acid sequence onlyD26096 (partial)D26099 (partial)D26098 (partial)M75731D26100D26102 (partial)

D26101 (partial)

CNCCCNCCCC

185363185185185

73 59 100 100 15477 58 77 61 185

18574 61 69 57 517

185

Nicotiana tabacum

Porphyra purpurea

Porphyra purpurea

L32 Astasia longaBrassica rapaEuglena gracilisLycopersicon esculentumMarchantia polymorphaNicotiana tabacumOryza sativaPorphyra purpureaVicia fabaZea mays

X16004, X75651Z26332X70810D17805 (partial)X04465Z00044 (as ORF55)X15901

C

C

C

C

C

C

C

C

C

C

X51471X64099

56 38 55 45 56848 15 85 83 58248 30 50 35 243

66843 18 74 61 18646 18 100 100 73347 22 67 56 26544 20 57 48 51752 26 83 68 25643 18 74 61 688

Cyanophora paradoxaEpifagus virginianaMarchantia polymorphaNicotiana tabacumOryza sativaPorphyra purpurea

X17498M81884X04465Z00044X15901

C 60 42 78 62 163C 52 37 93 85 712,714C 56 44 80 71 186C 52 37 100 100 560, 561C 60 42 82 73 265C 59 43 67 50 517


L23

L24

L25f

L28

L29

L30f

L31

X68078 N 57 36

C 51 24

C 60 37

731

517

517

MICROBIOL. REV.

L27

L33


TABLE 3. Chloroplast and cyanobacterial ribosomal proteins that have been identified by gene or protein sequence


%S %I %S %I

Zea mays X56673 C 57 38 81 75 686

L34 Cyanophora paradoxa C 49 40 598Porphyra purpurea C 60 49 517

L35 Cyanophora paradoxa X17063 C 55 38 61C 45c 69Porphyra purpurea C 52 36 67C 50c 517Spinacia oleracea M60449 N 64 42 100 100 577

L36 Astasia longa X16004, X75651 C 68 46 73 57 566Cryptomonas 'F X62348 (partial) C 124Cyanophora paradoxa C 78 62 92 76 598Epifagus virginiana M81884 C 81 57 95 92 712, 714Euglena gracilis X70810, Z11874 C 73 51 84 65 88, 243Marchantia polymorpha X04465 C 86 62 95 86 186Nicotiana tabacum Z00044 C 86 62 100 100 560, 561Oryza sativa X15901 C 89 68 95 92 265Pisum sativum Y00468, X15645 C 84 62 97 86 505, 506Porphyra purpurea C 81 59 95 70 517Spinacia oleracea X03496 C 84 62 97 95 571Zea mays M35956 C 89 68 95 92 402

CS-S5, PSrp-1, "S22" or "S30"g Spinacia oleracea X59270, X15344 N 28, 741Spinacia oleracea M55322 N 290

"S31" or SCS239" Spinacia oleracea 541, 674

PsCL189 Pisum sativum X14021 N 192

"L40999 Spinacia oleracea M58523 N 75

PsCL25M Pisum sativum X14022 N 192a Proteins Si through S21 and LI through 136 are named by reference to similar sequences in E. coli, and the location of the gene encoding them (N, nuclear; C,

chloroplast or cyanelle) is given for all eukaryotic species. A few additional chloroplast ribosomal proteins with no obvious similarity to E. coli proteins have beenidentified and appear at the end of the table.

b The percent similarity (%S) and percent identity (%I) to the E. coli and tobacco proteins or other reference land plant proteins were calculated by the gap routineof the Genetics Computer Group sequence analysis package.

c Tobacco sequence not available; spinach used instead for comparison.d The complete tobacco genome sequence (Z00044) has a stop codon in the terminal exon encoding the S12 protein, whereas the sequence by Fromm et al. (183)

(X03481) shows a full-length protein comparable to that from other chloroplast genes. The Swissprot sequence (P06309) omits the terminal residue (Tyr) of the firstexon of this protein. The composite sequence with these corrections made is identical to that for N. plumbaginifolia and was used for the comparisons given here.

I Tobacco sequence not available; pea used instead for comparison.fNo equivalent found so far in chloroplasts.g Proteins for which no equivalent appears to exist in E. coli.h Small, basic protein found in spinach ribosome preparations.

encoding this protein is absent from the completely sequencedchloroplast genomes of tobacco, rice, Marchantia polymorpha,and Euglena gracilis (243, 558) but appears in the Porphyrapurpurea chloroplast genome (514). A nuclear gene encodingthis protein has been identified in spinach (178, 179) andshown to have a light-independent, leaf-specific pattern ofexpression under the control of a negative nuclear factor, SlF,that down-regulates its promoter (740). Hahn et al. (240)reported that monoclonal antisera to E. coli Si reacted with achloroplast protein of spinach. Polyclonal antisera to twochloroplast-synthesized ribosomal proteins of C. reinhardtii("S-7" and "S-11" [547]) reacted with E. coli Si (510), as didantisera to mixed chloroplast ribosomal proteins of spinach(119). Subramanian et al. (608) also suggested the presence ofa chloroplast Si homolog in maize on the basis of affinity-binding experiments with a matrix-bound poly(U) column.

In E. coli the S2 protein interacts primarily with the 3'domain of the 16S rRNA in the 960 loop region and can becross-linked to proteins S3, S5, and S8 (500, 595, 599). The rps2gene encoding a ribosomal protein homologous to E. coli S2has been found in chloroplast genomes from diverse species(Table 3), typically mapping between the rpoB and rpoC genesencoding subunits of RNA polymerase and the atpI and atpHgenes encoding ATP synthase subunits. The deduced aminosequences of S2 proteins from land plants are highly conserved(Table 3), and those of three monocots (wheat, rice, andmaize) are nearly identical to one another. A nuclear DNAsequence in spinach with substantial homology to a portion ofthe chloroplast rps2 gene is thought to be an example of"promiscuous DNA," i.e., a DNA sequence found in more

than one genetic compartment (85).Protein S3, like S2, interacts with nucleotides in the 960 and

VOL. 58, 1994

724 HARRIS ET AL.

1050/1200 regions of E. coli 16S rRNA (465) and appears fromimmunoelectron microscopy studies of the 70S ribosome toreside near the site to which the polypeptide release factorRF-2 binds (632). The rps3 gene encoding the equivalentprotein has been sequenced from several chloroplast genomes(Table 3), where it has been found between rp122 and rp116, asis true for the corresponding genes in the S10 operon of E. coli(Fig. 5). In most algae and plants the rps3 gene is uninter-rupted, but in Euglena gracilis it contains two introns, one (102nt) belonging to the group III class (as defined by Christopherand Hallick [87]; also see reference 86) and the other (409 bp)being a "twintron" consisting of a 311-nucleotide group IIintron within a 98-nt group III intron (92). Splicing proceedssequentially, with the internal 311-nt intron being excised first.In the chloroplast genome of C. reinhardtii, there is no geneequivalent to rps3 in the expected location between rp122 andrp116 (277). However, Fong and Surzycki (172) found a longopen reading frame between the rpoB and rpoC genes, whose5' and 3' ends would encode a protein with substantialhomology to S3. The central portion of the predicted productof this open reading frame has no homology to S3, however,and the DNA sequence does not contain recognizable splicejunctions that would suggest that this region is in fact an intron.Liu et al. (366) found that this open reading frame is alsopresent in several other Chlamydomonas species. After trans-formation of C. reinhardtii cells with a construct containing thisopen reading frame interrupted by the bacterial aad antibioticresistance gene, the only resistant cells recovered were hetero-plasmic for the interrupted and native forms of the gene. Incontrast to transformants in which the same construct wasinserted into other regions of the genome, no homoplasmiccells containing only the interrupted gene could be obtained,strongly suggesting that this gene is not only functional but alsoessential to cell growth. No single transcript spanning thewhole gene could be detected, however, and the gene producthas not been identified (366).

In E. coli, S4 is one of the primary rRNA-binding proteinsthat initiate assembly of the 30S subunit (255, 465) and isassociated with the 5' domain of 16S rRNA, at a junction ofseveral helices. Together with S5 and S12, S4 participates in a

region designated by Oakes et al. (466) as the recognitioncomplex on the basis of its demonstrated involvement incodon-anticodon recognition and translational accuracy. Thisregion involves the 530 loop, the 900 loop region, and the 5'end of the 16S molecule which pairs with the region aroundresidue 912 (Fig. 3). Homologs of all three of these proteinshave been identified in yeast cytoplasmic ribosomes and ap-

pear to have similar functions (4). Mutations in the gene

encoding S4 in E. coli suppress streptomycin dependencemutations in the gene for S12 and increase translationalambiguity (ram mutants [7, 189, 335, 336, 475). The chloroplastgene encoding ribosomal protein S4 has been sequenced froma number of plants and algae (Table 3) and shows a highdegree of conservation in its first 25 amino acid residues and ina large block of approximately 120 residues in the centralportion of the protein. The C. reinhardtii S4 protein is some-what longer than all others examined so far, having twointernal insertions and a 22-amino-acid C-terminal extension(511).

In land plants, rps4 appears to be transcribed singly undercontrol of its own promoter and is not part of an operon withother ribosomal protein genes (608). In tobacco, rice, andMarchantia polymorpha, the rps4 gene is in the large single-copy region and is preceded by tRNAThr on the same strandand followed by tRNAser on the opposite strand. In C.reinhardtii, rps4 follows the large ribosomal protein operon

ending with rps8 (Fig. 5) but is separated from this operon byexon 1 of the psaA gene and two tRNA genes, tmM and trnG(277). In Euglena gracilis, rps4 is transcribed together withrpsll (242, 597). In the alga Cryptomonas 'F, rps4 is close torbcL on the same strand and is flanked by tRNAArg on theopposite strand (127), whereas in Cyanophora paradoxa,tRNA et and tRNAG'Y are adjacent to rps4 but on theopposite strand (32, 598).

In E. coli, the S5 protein is part of the recognition complex(466) and is the first protein of the small subunit whose crystalstructure has been determined (from B. stearothermophilus[507, 508]). The protein appears to be a somewhat elongatedmolecule with two distinct domains. Mutations affecting aminoacids 20 to 22 of E. coli S5 can confer spectinomycin resistance,whereas mutations at amino acids 104 and 112 have a ramphenotype and suppress streptomycin dependence mutationsin protein S12 (508). Some of the latter class of mutants arealso neamine resistant (721). These two conserved regions ofthe S5 protein are thought to be the sites of its interaction withrRNA (508). Genes encoding a protein with homology to E.coli S5 have been sequenced from the cyanelle genome ofCyanophora paradoxa (368, 423) and from the P. purpureachloroplast genome (517). No equivalent gene has been foundin any land plant chloroplast genome, however, nor does itappear in the chloroplast genome of Euglena gracilis.The S6 ribosomal protein of E. coli is implicated in mRNA

and tRNA binding and in termination (465, 632), and itappears to be a component of the platform region of the 30Sparticle (432, 622). A plastid equivalent of S6 is known so faronly from Porphyra purpurea (517).

In E. coli, protein S7 interacts with several clusters ofnucleotides in the 3' domain of 16S rRNA, in proximity to S9,S1O, and S19 (57, 136, 465, 499), and is one of the initiatingproteins of 30S assembly (255). Binding of S7 to 16S riRNA isa prerequisite to assembly of S9 and S19. As discussed above,the gene encoding S7 is transcribed together with that for S12in bacteria and in the chloroplast genomes of most plants andalgae examined (Fig. 5), the principal exceptions so far beingChlamydomonas species. In C. reinhardtii, the protein encodedby rps7 corresponds immunologically to the protein thatSchmidt et al. (547) identified as "S-20" (509). Althoughderived amino acid sequence identity between chloroplasts andbacteria is lower for S7 than for S12 (Table 3), antibodies to E.coli S7 do cross-react with a corresponding small-subunitprotein from spinach chloroplast ribosomes (18).

In E. coli, S8 is an RNA-binding protein that is essentialearly in assembly of the 30S subunit and interacts with a highlyconserved site in the central domain of 16S rRNA, designatedby Oakes et al. (466) as the platform ring (also see references141, 437, 465, and 622). It is associated with proteins S15 andS17 (57). It also has a key role in translational regulation of thespc operon in E. coli (719). S8 is moderately conservedphylogenetically and can be identified with equivalents ineukaryotic ribosomes (410, 708). The rps8 gene is chloroplastencoded (Table 3) and is one of at least three genes of thebacterial spc operon that remain linked in chloroplast genomes(Fig. 5). Most plastid S8 proteins have a central 4- to 7-amino-acid insertion compared with the E. coli protein, followed by ahighly conserved C-terminal region.

Protein S9 interacts with S7 and S19 in the 3' domain of theE. coli ribosome (57, 499). Bartsch (18) obtained cross-reac-tivity of antibody to E. coli S9 with a spinach chloroplastribosomal protein, but the gene encoding this protein has notbeen found in any of the land plant chloroplast genomes so farsequenced and is presumed to be nucleus encoded. However,an rps9 gene does appear in the chloroplast genomes of

MICROBIOLE REV.


Euglena (243), Porphyra (517), and Cryptomonas (122) speciesand in the cyanelle genome (598). Overall, the C-terminalportions of the S9 proteins appear more highly conserved thanthe N-terminal portions.

S10 is a late-assembly ribosomal protein of the 3' domain inE. coli 16S rRNA (465). A gene encoding a homolog ofS10 hasbeen found in the cyanelle genome (68, 451, 452), where itmaps downstream of the petFI gene. Sequences encoding anS10 protein have also been found in Cryptomonas D (122) andin Porphyra purpurea downstream of the tufA gene (517).

In E. coli, Sl1 interacts with S6 and S18 in the platformregion of the 30S subunit (466). S11 is highly conservedphylogenetically, with homologs also identified in eukaryoticribosomes (401, 708). There is some variability in lengthamong Sli proteins, localized to the N-terminal regions. Inchloroplast genomes of land plants, the rpsll gene is part ofthe large ribosomal protein operon that terminates in rpoA(Fig. 5). In Euglena gracilis, in which it is in a separate operonwith rps4, the rpsll gene contains two group III introns (597).

S12 appears to be the most highly conserved of all the smallsubunit proteins. The C. reinhardtii S12 protein is sufficientlysimilar to its bacterial counterpart that the rpsl2 gene from thisalga can be expressed in E. coli cells and the resulting proteincan assemble and function in the E. coli ribosome (364). In E.coli, S12 interacts with two regions of the 16S rRNA, the 530loop and 900 stem-loop. Mutations affecting either the S12protein or the 16S rRNA regions with which it associates canconfer streptomycin resistance in E. coli by reducing misread-ing induced by the drug (Table 2). Streptomycin resistancemutations have also been found at evolutionarily conservedsites in the S12 proteins or 16S rRNA of Chlamydomonasspecies, Euglena gracilis, and tobacco, and streptomycin depen-dence mutations affecting the S12 protein have been found inbacteria and in C. reinhardtii (Table 2). Streptomycin-depen-dent E. coli mutants exhibit hyperaccurate proofreading andreduced efficiency of binding of EF-Tu (25, 102, 150).

In E. coli, S13 appears to be located at the head of the 30Ssubunit, near the center of the surface that faces the 50Ssubunit (432). It has been reported to cross-link to S7 and S19in the 3' domain of the 16S rRNA (599). Genes encoding acognate protein have been identified in the cyanelle genome(598) and in the Porphyra chloroplast genome (517) but haveso far not been reported to occur in other algae or land plants.The late-assembly ribosomal protein S14 interacts with the

3' domain of 16S rRNA in E. coli (465). The chloroplast-encoded Euglena rpsl4 gene is part of the large ribosomalprotein cluster as it is in E. coli, downstream from rp136 and atRNAIle gene (457), whereas in other chloroplast genomes it isfound outside this cluster. In land plants, the rpsl4 gene islocated in the chloroplast genome downstream from the psaAandpsaB genes encoding reaction center proteins of photosys-tem 1 (81, 82, 307, 496, 560). In Cyanophora paradoxa, the rpsl4gene is upstream of open reading frame ORF512 andpsaA andis transcribed divergently from these two genes (598). InPorphyra purpurea, rpsl4 is flanked by petF and petG (514).

S15 is an early-assembly protein in E. coli that interacts withthe central domain of the 16S rRNA, together with S6, S8, andS18 in the platform ring (57, 151, 438, 465, 466, 622). Intobacco and liverwort, the rpsl5 gene is in the small single-copyregion of the chloroplast genome (312, 560), whereas in threemonocots (rice [265], rye [501], and maize [170]) it is in theinverted repeat, very close to the boundary with the smallsingle-copy region. This gene is missing from the chloroplastgenomes of Euglena gracilis (243) and Porphyra purpurea (517)and from the cyanelle genome (32, 598).

In E. coli, S16 is a protein associated with the 5' domain of

the 16S rRNA and is probably close to S4 in the assembled 30Ssubunit (594). Montesano-Roditis et al. (432) have localizedS16 to the 30S body near its junction with the platform, on thesurface facing the 50S particle. In various angiosperms, S16 isencoded by a chloroplast gene (Table 3), but the rps16 gene isabsent from the Marchantia chloroplast genome (471). It isfound, however, in the plastid genome of the red alga Cya-nidium caldarium (385). In tobacco, the rpsl6 gene has an860-bp intron with boundary sequences similar to the introns inrpsl2, rp12, and several tobacco tRNA genes (559). The rpsl6genes of mustard (450), barley (556), and maize (293) alsocontain introns, but that of Cyanidium caldarium is uninter-rupted (385).

S17 is one of the primary assembly proteins in E. coli andbinds to 16S rRNA in the 5' domain (213, 255, 465, 594). It isone of only three chloroplast ribosomal proteins of the smallsubunit to date for which a nucleus-encoded gene has beencloned and sequenced (Table 3) (606). This protein contains ahighly conserved region which can be identified in bothbacterial (S17) and eukaryotic (Sli) ribosomal proteins. Com-parison of the deduced amino acid sequences of the equivalentchloroplast S17 and cytosolic Sli ribosomal proteins fromArabidopsis thaliana with E. coli S17 supports the notion thatchloroplast S17 is derived from a prokaryotic endosymbiontand not from duplication of the eukaryotic S11 gene (196). Thepresence of an rpsl7 gene in the cyanelle genome of Cyano-phora paradoxa (368) and in the chloroplast genome of Por-phyra purpurea (514) is consistent with this hypothesis.

Proteins S18 and S6 assemble coordinately in the bacterialribosome to form part of the platform ring in the centraldomain (432, 466, 622). The chloroplast gene encoding S18 hasbeen found to be part of an operon with rp133 in the completelysequenced chloroplast genomes of tobacco, rice, and Mar-chantia polymorpha, as well as in Cyanophora paradoxa, but itis absent from this operon in Euglena gracilis (Fig. 5). Theangiosperm S18 proteins have N-terminal extensions com-pared with E. coli, Marchantia polymorpha, and Cyanophoraparadoxa, containing various numbers of repeats of a hydro-philic heptapeptide (686). A C-terminal extension found in theS18 proteins of rice and maize is missing from this protein intobacco (561, 686).The S19 protein of E. coli interacts with proteins S7, S9, and

S14 and with several helices in the 3' domain of the 16S rRNAmolecule (57). The gene encoding S19 was the first chloro-plast-encoded ribosomal protein gene to be identified andsequenced (612, 745). In plants with the "typical" chloroplastinverted-repeat structure, the rpsl9 gene and the adjacent rp12and rp122 genes are located at or near the boundary betweenthe inverted repeat and the large single-copy region. In to-bacco (560) and Marchantia polymorpha (186), rpsl9 is entirelywithin the large single-copy region but near the inverted-repeatjunction, whereas in rice (433) the whole gene is within theinverted repeat. Zurawski et al. (746) found that the first 48codons of rpsl9 in spinach were in the inverted repeat, with 44codons homologous to the 3' end of the E. coli gene beingpresent only on one side of the large single-copy region.Thomas et al. (635, 636) showed that this complete copy of thegene was expressed, whereas the rpsl9' sequences beginning inthe other side of the inverted repeat and extending for 66codons into the adjoining unique sequence region (745) werenot transcribed. The rpsl9 gene also straddles the boundary ofthe inverted repeat in Spirodela oligorhiza (494) and in mustard(454).

S20, which was also identified in early ribosome studies in E.coli as L26, is a primary RNA-binding protein that interactswith the 5' domain of the 16S rRNA (255,594). Deletion of the

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726 HARRIS ET AL.

rpsT gene encoding S20 in E. coli results in increased misread-ing of all three nonsense codons and a deficiency in assemblyof 30S and 50S subunits to form 70S monomers (534). An rps2Ogene has been identified in the cyanelle genome (32) and in thePorphyra chloroplast (517).

E. coli S21 interacts with the central domain in the platformring (466). No equivalent protein has been identified so far inthe chloroplast ribosome.

Proteins of the Large Subunit

In E. coli, ribosomal protein Li forms a prominent ridge onthe large subunit (177, 466,599) and has been demonstrated tobind to nt 2100 to 2200 on theE. coli 23S rRNA, a region thatshows a high degree of conservation among chloroplast 23Ssequences. The gene encoding this protein is present in thecyanelle genome (32) and in the Porphyra chloroplast genome(517) but is absent from the completely sequenced plastidgenomes of land plants. Antibodies to E. coli Ll cross-reactedwith a spinach chloroplast ribosomal protein (8). cDNAsencoding chloroplast Li have been cloned from the nucleargenomes of pea, spinach, and Arabidopsis thaliana, and thenuclear gene has been isolated and characterized from Arabi-dopsis thaliana (300).

In E. coli, the L2 protein binds to domain IV of the 23SrRNA molecule, and cross-links specifically to nt 1818 to 1823(744), in a stem-loop structure that is part of the peptidyltrans-ferase center and is conserved in chloroplast 23S rRNAs.Site-directed mutagenesis of a conserved region in the E. coliL2 protein outside the 23S binding site has been used toproduce temperature-sensitive mutants that are impaired inassembly of the 50S subunit (526). The L2 protein is encodedin the chloroplast genomes of all land plants and algae so farexamined (Table 3). The L2 protein itself is moderatelyconserved, and its equivalent has been identified in eukaryoticribosomes (708). The C. reinhardtii protein "L-1," which issynthesized in the chloroplast (547), appears to be encoded bythe rpl2 gene since "L-1" antibodies cross-react with E. coli L2(510). Kamp et al. (292) showed that the N-terminal aminoacid of the L2 protein in spinach is N-methylalanine, the firstdemonstration of methylation of a chloroplast ribosomal pro-tein. Several ribosomal proteins ofE. coli are methylated, butL2 is not among these. The maize rpl2 gene begins with anACG codon, which is edited to AUG at the transcript level(321). The 3'-terminal ends of the deduced L2 amino acidsequences for spinach and Nicotiana debneyi published byZurawski et al. (745) appear to lack homology to the corre-sponding regions from other chloroplast and bacterial L2proteins. However, if a single-base insertion is made after theamino acid 226 of the spinach gene (changing the sequenceCCC ACG GGG GTG GTG ....[Pro Thr Gly Val Val.1..].. toCCN CAC GGG GGT GGT ....[Pro His Gly Gly Gly... .], thereading frame is shifted to specify 45 additional amino acidsthat resemble the consensus sequence much more closely. Thecorresponding change in the N. debneyi sequence produces a Cterminus identical to that of N. tabacum as determined byShinozaki et al. (561). The rp12 genes of tobacco, spinach, rice,and maize are located in the inverted-repeat region, but thoseof Marchantia polymorpha and C. reinhardtii are in single-copyDNA. The spinach, C. reinhardtii, and Euglena genes areuninterrupted, whereas those of many other land plants con-tain a single group II intron (133). The intron insertion sitesare identical in the Nicotiana, rice, and Marchantia genes, andthe introns themselves have a high degree of nucleotidesequence identity. Downie et al. (133) determined the distri-bution of the rpl2 intron in 390 species from 116 angiosperm

families and documented six independent losses of this intronamong dicotyledons.The L3 and L4 proteins of E. coli both bind to the 23S rRNA

molecule and have been identified with analogous proteins inarchaebacterial and eukaryotic ribosomes (215, 708). Genesencoding an L3 protein have been sequenced from the Cyano-phora cyanelle genome (161) and from the Porphyra chloro-plast genome (517). Neither an rp13 nor an rpl4 gene has beenfound in the completely sequenced chloroplast genomes oftobacco, rice, Marchantia polymorpha, or Euglena gracilisBartsch (18) found a cross-reaction between antibody toE. coliL3 and a spinach chloroplast ribosomal protein which has notbeen further characterized.Genes encoding a protein corresponding to the 5S RNA-

binding protein LS of E. coli have been found in the Euglena,C. reinhardtii, Porphyra, Astasia, and Cyanophora plastid ge-nomes (Table 3). However, no equivalent gene has been foundin any land plant chloroplast genome. Antibody to Chlamydo-monas chloroplast ribosomal protein "L-13" (547) reacts withE. coli LS and with a ribosomal protein ofAnabaena sp. (510).A weak reaction was also seen to a spinach protein ("LIO"),whose site of synthesis is uncertain (121). The Chlamydomonas"L-13" protein is known to be synthesized in the chloroplast(547), suggesting that this is the product of the chloroplast-encoded rplS gene sequenced by Huang and Liu (277).TheE. coli L6 protein binds to domain VI of 23S rRNA (90).

Genes encoding an equivalent protein have been found in thecyanelle genome of Cyanophora paradoxa (69) and the Por-phyra chloroplast genome (514).

The protein originally identified as L7 in E. coli is in fact theaminoacetylated form of L12, and L8 is a complex of L7/L12and L10 (358).

Protein L9 of E. coli is an elongated protein with distinctterminal domains which is associated with the protuberanceformed by protein Li and the region of the 23S rRNA to whichit binds (57, 213, 266). Genes encoding a protein equivalent toE. coli L9 have been sequenced from the nuclear genomes ofpea (192) and Arabidopsis thaliana (640) and from the Por-phyra chloroplast genome (517). The E. coli L9 protein cross-reacts slightly with antibody to the acidic chloroplast ribosomalprotein "L-30" from C. reinhardtii (510).

E. coli protein L10 (L8) forms the base of the ribosomalstalk in a pentameric complex with two dimers of L7/L12 and,like L7/L12, appears to be a universal constituent of eubacte-rial, eukaryotic, and archaebacterial ribosomes (708). A cya-nobacterial gene encoding this protein has been found (539,564) but no chloroplast equivalent has yet been identified.Presumably, chloroplast L10 is encoded by a nuclear gene inplants.L1i is an early-assembly protein that constitutes part of the

GTPase center in theE. coli ribosome (528) and also has beenidentified in eubacterial, eukaryotic, and archaebacterial ribo-somes (708). Nuclear genes encoding a chloroplast L1 ho-molog have been cloned from spinach andArabidopsis thaliana(543, 579, 606), while plastid genes have been identified inPorphyra purpurea (517) and Cyanophora paradoxa (32). InE.coli, L1i has the most extensive posttranslational modification(nine methyl groups) of all ribosomal proteins (708); the samemodifications occur in the spinach chloroplast Lii in thecorresponding amino acid residues, located in conserved se-quence contexts (607). Mutations in Bacillus megaterium andB. subtilis conferring resistance to the antibiotic thiostreptoncause the loss of Lii from the ribosomes (16, 101). Lli doesnot bind thiostrepton itself in solution but enhances thiostrep-ton binding to 23S rRNA (102, 639). Bacterial thiostrepton-resistant mutants with altered 23S rRNA have also been found

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(149, 533). McElwain et al. (413) have isolated a thiostrepton-resistant mutant of C. reinhardtii whose chloroplast ribosomesare resistant to the drug in vitro. The large subunits of themutant ribosomes lack a cytoplasmically synthesized protein("L-23") that, on the basis of size and immunological criteria,appears to be the equivalent of the E. coli L1i protein. Inpulse-labeling experiments, this Chlamydomonas mutant syn-thesizes small amounts of protein "L-23," but the protein failsto assemble into chloroplast ribosomes. The mutation showsMendelian inheritance, suggesting that rplll is a nuclear genein C. reinhardtii.The acidic protein L7/L12 is one of the most intensively

studied ribosomal components (57, 474, 708). Four L12 mol-ecules are present in each ribosome of E. coli, together withone L10 polypeptide, forming the stalk of the 50S subunit andinteracting with elongation factor EF-G which binds near thebase of the stalk. The homologous protein was identified inspinach chloroplasts by direct sequencing of tryptic peptides(19) and was predicted to have a tertiary structure similar tothat of its counterpart in E. coli (271, 345). cDNA clonesencoding this protein have been isolated and sequenced fromthe nuclear genomes of several land plants (Table 3), and thegene has been cloned and characterized from Arabidopsisthaliana and spinach (689). In Arabidopsis thaliana (689), L12is encoded by a multigene family with one silent and twofunctional genes, the functional genes both being closely linkedto cytosolic tRNA genes (this is the first such case identified fora chloroplast ribosomal protein [607]). However, L12 is en-coded by a chloroplast gene in Euglena gracilis and by acyanelle gene in Cyanophora paradoxa. The derived amino acidsequence of the cloned spinach gene includes three aminoacids that were apparently overlooked in the primary sequenceof the corresponding protein published by Bartsch et al. (19).Sibold and Subramanian (564) have compared the spinach andbacterial sequences with the L12 protein of the cyanobacte-rium Synechocystis sp. Like the E. coli protein, spinach L12 ispresent in multiple copies per ribosome, but it lacks theN-terminal acetylation seen in the bacterial protein (19).

Giese and Subramanian (202) reported that the transitpeptide sequence of a spinach gene for L12 contains two ATGcodons, each in a consensus initiation context, that would yieldthe same mature peptide after transport into chloroplast andN-terminal cleavage. Genes for L13, L35, and the novelprotein Psrp-1 have similar duplicated ATGs. Experiments inwhich the 5' part of the L12 gene was fused to a reporter genedemonstrated that both codons can be used in vitro and inspinach protoplasts, with about 25% of initiations occurring atthe second codon. Such an arrangement may enhance transla-tional efficiency. However, Elhag et al. (155) found that bothL12 cDNAs from tobacco had only a single ATG, correspond-ing to the first ATG of the spinach gene.The L13 protein of E. coli interacts with the 5' domain of the

23S rRNA molecule, in proximity to L4, L21, L28, and L29(57). A nuclear cDNA clone encoding a chloroplast ribosomalprotein equivalent to E. coli L13 has been identified in spinach(490, 609), and a chloroplast gene for this protein has beenfound in Porphyra purpurea (517). The spinach protein has54% deduced amino acid identity with that of E. coli over the142 amino acid residues that can be aligned, but it is precededby 52 residues at the N terminus with no homology to anyknown protein. Upstream of this sequence are 47 amino acidswhich appear to be a transit peptide. The chloroplast proteinalso has a C-terminal extension with no homology to E. coliL13. However, spinach L13 translated in E. coli from cDNAconstructs was found to be incorporated into functional ribo-somes (203). The N-terminal extension was removable by mild

protease digestion, suggesting that it remained on the ribo-some surface (203).L14 in E. coli is a late-assembly protein and does not bind

directly to 23S rRNA (458). Antibody-binding studies suggestthat in B. stearothermophilus this protein is located on thesurface of the 50S subunit (599). Genes encoding proteins witha high degree of homology to E. coli L14 have been found inchloroplast genomes of all plants and algae examined, exceptin the parasitic plant Epifagus virginiana, in which a pseudo-gene is present instead (714) (Table 3).

In E. coli, both L15 and L16 are late-assembly proteins thatare associated with the peptidyltransferase center but seem tobe nonessential for ribosome function. Fully active ribosomeslacking both these proteins, as well as L30, can be reconstitutedin vitro by modifying the conditions of the reconstitutionprocedure (175). Nuclear genes encoding chloroplast ribo-somal protein L15 have been reported from Arabidopsis thali-ana and pea (640), and no rpll5 sequences have been identifiedin any chloroplast genome to date (Table 3). The spinachchloroplast homolog of L15 is significantly larger than itscounterpart in E. coli, owing to extensions at the N-terminaland probably also the C-terminal ends (291).

In contrast to L15, ribosomal protein L16 is encoded by achloroplast gene located in a conserved operon in all speciesexamined (Table 3; Fig. 5). In land plants, the rpll6 gene is splitinto two exons (e.g., 498), whereas in Chlamydomonas rein-hardtii (373) and Gracilaria tenuistipitata (294), it is uninter-rupted. The Euglena gene contains three introns (86, 93).Antibodies to a chloroplast-encoded Chlamydomonas protein("L-17") cross-react with E. coli L16, with spinach "L24," andwith an Anabaena protein that comigrates with E. coli L16(510).

Protein L17 of E. coli has been shown to bind 23S rRNA(358). A mitochondrial homolog has been identified in Sac-charomyces cerevisiae (708), but no corresponding chloroplastprotein has been found. However, proteins of similar chargeand size were seen on two-dimensional electrophoresis ofribosomal proteins from C. reinhardtii and Anabaena sp. (510).Antibodies to E. coli L17 were also observed to cross-react witha chloroplast ribosomal protein from spinach (18).

Protein L18, which is highly conserved in bacteria, binds to5S rRNA and is associated with the peptidyltransferase center(91). Genes encoding a ribosomal protein equivalent to E. coliL18 have been found in the cyanelle genome of Cyanophoraparadoxa (423) and in the Porphyra chloroplast genome (517).Spinach ribosomal protein "CS-L13" is a homolog of E. coliL22 (see below), with N- and C-terminal extensions that haveno sequence homology with the 5S-binding proteins of E. colibut show some structural similarity to L18 (651).A gene encoding a protein equivalent to E. coli L19 has been

found in the Porphyra chloroplast genome (514) and in thecyanelle genome (598). Little is known about the function ofthis protein in either bacterial or chloroplast ribosomes.An equivalent of the early-assembly, RNA-binding protein

L20 of E. coli is encoded in chloroplast genomes of plants andalgae and in the cyanelle genome of Cyanophora paradoxa(Table 3).

Protein L21 of E. coli can be cross-linked to the 5' domainof the 23S rRNA molecule and, together with L4, may have a

second contact to the 23S molecule in the adjacent domain (57,481). The gene encoding L21 is chloroplast encoded in Mar-chantia polymorpha and in the red algae Cyanidium caldariumand P. purpurea (Table 3) but is absent from the chloroplastgenomes of rice, tobacco, and Euglena gracilis. A spinachnuclear gene encoding L21 has been found to contain fourintrons in its central region (340, 578). Two transcription start

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728 HARRIS ET AL.

sites were identified, one which appears to be constitutive andthe other which appears to be induced only in leaf tissue (340).The spinach L21 protein (formerly "CS-L7" [378]) is consid-erably longer than its homologs from E. coli and Marchantiapolymorpha, having extensions at both the N and C termini.The carboxyl-terminal extension contains seven Ala-Glu re-peats, creating a region of high negative charge, and theprotein as a whole is acidic, in contrast to E. coli L21, which isbasic. However, this protein can be incorporated into E. coliribosomes assembled in vivo (71, 685). The spinach L21protein shows greater homology to E. coli L21 than it does tothe chloroplast-encoded Marchantia protein, prompting Mar-tin et al. (408) to hypothesize that spinach L21 arose either byduplication of a nuclear gene for a corresponding protein ofthe cytoplasmic ribosome or by transfer of a mitochondrialgene, rather than by transfer of a chloroplast gene to thenucleus. A mitochondrial origin seems unlikely, since no geneencoding the equivalent of L21 has so far been identified in a

mitochondrial genome of any plant (371).Protein L22 binds to 23S rRNA early in assembly of the 50S

particle in E. coli and is one of only five proteins both necessary

and sufficient to formation of the core precursor particle RI*(459). Equivalent proteins have been identified in archaebac-teria and cytoplasmic ribosomes (381, 708). The rp122 gene isfound in chloroplast genomes of all land plants so far exam-

ined, with the exception of two unrelated groups of angio-sperms, the legumes and the parasitic plant Epifagus virginiana(193, 485) (Table 3). A nuclear gene encoding L22 has beencloned from pea (193). In this gene, the exon encoding theputative N-terminal transit peptide is separated by an intronfrom the conserved structural gene. Gantt et al. (193) specu-

lated that the transit peptide sequence may have been acquiredby a form of exon shuffling. The rp122 gene is also missing fromthe relic of the S10 operon in the C. reinhardtii chloroplastgenome (277) but is found in the expected location in theplastid genomes of Euglena gracilis and the red algae Gracilariatenuistipitata and Porphyra purpurea (86, 295, 514) and in thecyanelle genome (163, 423).The spinach rp122 gene encodes a protein with a central

region homologous to all L22 proteins but has N-terminal andC-terminal extensions with structural similarity to the E. coliL18 and L25 proteins on the basis of hydropathy profiles (651,741). The spinach L22 protein binds to SS rRNA, protectingthree nonoverlapping binding sites (76, 651). In E. coli, how-ever, L22 does not bind SS rRNA but L18 and L25 do. Theseobservations suggest the interesting possibility that the spinachL22 protein ("CS-L13") serves the composite functions of allthree of these proteins. Carol et al. (76) have shown that theL22-like central domain of the spinach protein is required forSS binding, so that this domain appears to have a functionlacking in the E. coli protein. However, both the spinach and E.coli L22 proteins bind erythromycin. The monocots rice andmaize have L22 proteins with similar 29-residue N-terminalextensions, which, however, have little homology to the spinachextension. N-terminal extensions are lacking in the L22 pro-

teins of tobacco, Marchantia, Euglena, and Gracilaria species;the tobacco protein has a C terminus that is longer than that ofMarchantia polymorpha but considerably shorter than that ofspinach.The position of L23 in the E. coli ribosome has been

controversial, with cross-linking and immunoelectron micros-copy studies giving conflicting results (91, 447). Kruft et al.(328) propose that it has an elongated structure, with theN-terminal domain close to L29 at the base of the 50S subunitand the C-terminal domain on the ribosomal surface close to

the peptidyltransferase center. Proteins equivalent to E. coli

L23 are found in ribosomes from eubacteria, organelles,archaebacteria, and cytoplasmic ribosomes of eukaryotes (513,708). The equivalent cytoplasmic ribosomal proteins (calledL25 proteins) have an extended amino terminus and a carboxyterminus that resembles the archaebacterial L23 protein morethan the eubacterial one.

Sequences with relatively low homology to the gene encod-ing E. coli L23 have been found in the "S1O"-like operons inchloroplast genomes of a number of plants (Table 3; Fig. 5).While these rp123 sequences are in the same position as theE.coli gene for L23 in this operon, not all the chloroplastsequences form continuous open reading frames and somemay be pseudogenes (45, 732, 746). The rp123 genes of spinachand four related dicots appear to have sustained a 14-bpdeletion approximately in the center of the coding sequence,creating two overlapping open reading frames with homologyto the two halves of the tobacco gene (635, 746). Transcriptsfor both reading frames could be detected in vivo by S1mapping in spinach (635). However, no radioactive peptidescorresponding to these transcripts were seen on two-dimen-sional electrophoresis of the products of a coupled transcrip-tion-translation system. Furthermore, when chloroplast ribo-somal proteins of a size close to those expected for thechloroplast rpl23 gene products, either singly or spliced, weresubjected to N-terminal sequencing, none of the sequencesobtained corresponded to the predicted sequence of the splitrp123 gene. Bubunenko et al. (70) have recently reported thatchloroplast ribosomes of spinach contain no protein thatcross-reacts with the product of the functional chloroplast rpl23gene of maize but do contain a protein with strong homologyto the L23 equivalent of eukaryotic cytoplasmic ribosomes.This is the first suggestion that a nuclear gene encoding acytoplasmic ribosomal protein has been substituted for anonfunctional chloroplast gene.

In Epifagus virginiana the plastid rpl23 sequence is also apseudogene (714). However, Yokoi et al. (732) found that thetobacco rpl23 gene, which does have a continuous open readingframe, appears to be functional, since the N-terminal sequenceof a 13-kDa protein from the 50S ribosomal subunit exactlymatches that predicted from the chloroplast rp123 gene. Therp123 genes from three monocots (rice, wheat, and maize) arealso uninterrupted, and their derived amino acid sequences arevirtually identical to one another. In rice an rp123 gene islocated in the inverted repeat, but an open reading frame withhomology to rpl23 is also present in the large single-copyregion between rbcL and petA (265). Analysis of the corre-sponding region in wheat (Triticum aestivum) and two closelyrelated plants,Aegilops squarrosa andA. crassa, has revealed anapparent rp123 pseudogene in wheat andA. crassa but not inA.squarrosa, whose chloroplast genome seems to have sustaineda deletion in this region as a result of illegitimate recombina-tion between short direct repeats (45, 46, 469). Wheat has alength polymorphism just downstream of this gene comparedwith A. crassa, also apparently the result of an illegitimaterecombination event between relics of short repeats. Se-quences with strong homology to the chloroplast rpl23 genesand pseudogenes have also been detected in mitochondrialDNA of rice and maize (45).The rpl23 gene is missing from the corresponding operon of

the cyanelle genome (32, 598). In Euglena gracilis, the rp123gene is in the expected position at the start of the "S10" operonbut is interrupted by three group III introns (243). An unin-terrupted rp123 gene has been sequenced from the chloroplastgenome of C. reinhardtii, in the expected position at the start ofthe "S10" operon (277). However Randolph-Anderson et al.(510) found that antibody to C. reinhardtii "L-29," a cytoplas-

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mically synthesized protein, cross-reacts with E. coli L23 andwith spinach ribosomal protein "L28." This antibody showedno cross-reaction with Anabaena "L24," a protein whichcomigrates with E. coli L23 on two-dimensional gels.L24 is an early-assembly rRNA-binding protein in bacteria,

which, together with L3, is essential for initiation of assemblyof the 50S subunit but seems not to be essential either for latestages of ribosome assembly or for translation in vivo. An E.coli mutant lacking L24 can grow, albeit very slowly (257, 461).A reading frame specifying L24 has been found in the chloro-plast genome of P. purpurea, in the position expected based onsimilarity to the E. coli spc operon (514), but this proteinappears to be nucleus encoded in land plants, and cDNAsencoding it have been sequenced from pea (192), spinach (75,339), and tobacco (153). The plant sequences have transitpeptides of about 70 amino acids, as well as highly conservedC-terminal extensions.L25 is a 5S-binding protein in E. coli (57). No exact homolog

has been identified in chloroplast ribosomes. However, inspinach two chloroplast ribosomal proteins, L22 and "CS-12,"have been shown to bind SS rRNA and may thus together servethe same function as E. coli L5, L18, and L25 (76, 651) (seeabove). The 5S-binding domain of spinach "CS-12" showsstructural similarity to that of L25.The protein formerly designated as L26 of E. coli is now

identified as S20 (706).L27 is a conserved protein that maps by immunoelectron

microscopy to the base of the central protuberance of the 50Ssubunit of the E. coli ribosome and appears to be associatedwith the peptidyltransferase center (57, 744). Plastid genesencoding this protein have been sequenced from chromophyteand rhodophyte algae (185, 517), but the gene appears to benucleus encoded in green algae and in land plants. Two cDNAsencoding a protein homologous to E. coli L27 have beensequenced from tobacco (154) and found to have differing3'-flanking sequences, suggesting that the tobacco nucleargenome encodes more than one L27 gene. The identity ofthese cDNAs was confirmed by comparing the predicted aminoacid sequences with that determined for the purified L27protein (154). N-terminal amino acid sequencing of the cyto-plasmically synthesized ribosomal protein designated "L-18" inC. reinhardtii (547) indicates that this protein is the E. coli L27homolog (363), but the gene has not yet been cloned. Schmidtet al. (545, 546) found that C. reinhardtii "L-18" is synthesizedas an 18.5-kDa precursor that undergoes a two-step processingreaction. Conversion of the 17-kDa intermediate identified inpulse-labeling experiments to the mature 15.5-kDa form re-quires chloroplast protein synthesis. Liu et al. (363) showedthat the 17-kDa intermediate specifically associates with aribosomal complex that migrates with the ribosomal largesubunit before being processed to the mature protein. Thissuggests that the second processing step may be required formaturation of the 50S ribosomal subunit. Antibody to C.reinhardtii "L-18" cross-reacts with E. coli L27, with spinach"L22" (terminology of Mache et al. [378]), and with anAnabaena protein ("L23" [510]). Elhag and Bourque (154)show the alignments of the tobacco L27 sequence with thepartial L27 sequences of C. reinhardtii (547) and spinach (607),and with that of the yeast mitochondrial ribosomal proteinMRP7 (167). Elhag and Bourque (154) note that this is the firstexample of a chloroplast ribosomal protein for which thesequence of a presumably homologous mitochondrial ribo-somal protein is known.

L28, which cross-links to the 5' domain of the 23S rRNA, isadded to the E. coli ribosome relatively late in assembly (57,459), and mutants lacking L28 are viable although cold sensi-

tive (103). A nuclear gene encoding a protein with sequencesimilarity to E. coli protein L28 has been isolated from tobacco(731). The "L-31" protein of C. reinhardtii chloroplast ribo-somes was observed to comigrate with E. coli L28, but immu-nological cross-reactivity was not tested (510).

E. coli protein L29 also can be cross-linked to the 5' domainof the 23S molecule (57) but associates with the ribosomalparticle early in the assembly process (459). L29 appears in thesame ribosomal neighborhood as proteins L2, L4, L15, andL34 (676). A gene encoding an equivalent chloroplast proteinhas so far been found only in Porphyra purpurea (514).

Protein L30 of E. coli assembles late and can be eliminatedby mutation (103, 459). An equivalent protein has beenidentified in archaebacteria (708) but so far not in chloroplasts.L31 is a late-assembly protein in E. coli (358, 459) and is knownso far only from the Porphyra chloroplast genome.

E. coli L32 also associates with the ribosome late in theassembly process (459). Genes encoding L32 have been foundin the chloroplast genomes of several plants and algae (Table3). However, this gene is missing from the Epifagus chloroplastgenome (714). Deduced amino acid similarity to the E. coligene is low (Table 3), but hydropathy plots suggest that theplant and bacterial proteins are similar in conformation (733).The N-terminal portions of the chloroplast L32 proteins arehighly conserved in amino acid sequence, whereas the C-terminal ends are variable in sequence and in length.The E. coli L33 protein can be cross-linked to 23S rRNA at

positions 2422 to 2424 (481) and to proteins Li and L27 (676).Mutants of E. coli lacking L33 are viable but cold sensitive(103). The rp133 gene is chloroplast encoded in land plants andPorphyra purpurea and has also been sequenced from thecyanelle genome (Table 3) but is missing from the Euglenachloroplast genome (243).

Protein L34 has been characterized in a number of eubac-terial species but has not yet been identified in chloroplastsexcept for those in Cyanophora and Porphyra purpurea. In theE. coli ribosome, it is found in a neighborhood with proteinsL2, IA, L15, and L29 (676).

Protein L35, formerly designated ribosomal protein A in E.coli (675), is encoded in the nuclear genome of spinach (577)but in the chloroplast genome of P. purpurea (514) and in thecyanelle genome of Cyanophora paradocxa (69).

L36, the product of the E. coli gene formerly designated secX(675), is chloroplast encoded in all plants and algae so farexamined (Table 3). The high degree of conservation of theamino acid sequence of the chloroplast L36 proteins comparedwith E. coli (Table 3) suggests that this small protein may havean important but as yet unknown role in the ribosome.

Chloroplast Ribosomal Proteins with No ObviousHomology to Those of E. coli

Zhou and Mache (741) reported that spinach chloroplastscontain relatively large amounts of a unique ribosomal protein,"CS-S5." The deduced amino acid sequence of a full-lengthcDNA clone for the nuclear gene encoding this ribosomalprotein shows no sequence similarity to any bacterial ribo-somal protein (28, 741). Working independently, Johnson et al.(290) characterized a 26-kDa protein from spinach chloroplastribosomes ("PSrp-1"), which appears to be identical to "CS-SS." The DNA sequences determined by the two groups differin three nucleotides, one of which creates a frame shift thatchanges the predicted C-terminal sequence. Direct sequencingof protease-generated internal peptides supports the 236-amino-acid sequence published by Johnson et al. (290). Theprecursor form of this protein contains 302 amino acids.

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730 HARRIS ET AL.

Lagrange et al. (339) proposed to designate this ribosomalprotein S22, since S21 is the highest-numbered protein of theE. coli small subunit. Schmidt et al. (541) have proposedalternatively that the numbers 22 to 29 be skipped and that thisprotein be named S30 instead.

Schmidt et al. (541) and Wada et al. (674) have indepen-dently identified another novel protein in preparations ofspinach chloroplast ribosomes. Schmidt et al. described a basicprotein of about 7.5 kDa and gave it the designation S31. Asequence of 43 of the estimated 60 amino acids constitutingthis protein showed no homology to any known E. coliribosomal protein or to any other sequence available in publicdatabases, nor did it correspond to the derived amino acidsequence of any coding region in a published chloroplast DNAsequence. However, the sequenced region was shown to have42% identity to the unpublished sequence of a small basicprotein isolated from the bacterium Thermus thermophilus.Wada et al. (674) described a 5-kDa protein, SCS23, with noapparent homology or immunological cross-reactivity to any E.coli ribosomal protein. The N-terminal sequence of this pro-tein is similar to that published by Schmidt et al. (541).Two cDNAs isolated from pea encode ribosomal proteins of

moderate size with no recognizable similarity to any ribosomalprotein of E. coli (192). The PsCL18 gene encodes a protein of145 amino acids, including a transit sequence of approximately50 amino acids, and the PsCL25 gene specifies a protein of 104amino acids, of which about 30 amino acids constitute a transitsequence. Typical of ribosomal proteins, the deduced aminoacid sequences of both PsCL18 and PsCL25 have a highcontent of lysine and arginine residues, and a consequent highnet positive charge, but differ in the distribution of thesecharged amino acids. PsCL18 has a highly charged, highly basiccarboxyl end, whereas the carboxyl terminal of PsCL25 con-tains mostly uncharged amino acids with four aspartic acidresidues constituting the only charged species. No E. coliribosomal protein has a carboxyl terminus resembling either ofthese nucleus-encoded chloroplast ribosomal proteins frompea. A protein similar to PsCL18 has been isolated fromspinach and designated L40 (75, 339). This protein appears tobe encoded by a single-copy nuclear gene and to contain 142amino acids with 54% sequence identity to pea CsL18. This isa slightly lower sequence identity than is seen between othernucleus-encoded ribosomal proteins of higher plants, e.g., S17,L12, and L15 (Table 3).

Comparative Analysis of Ribosomal Proteins

Sequence comparisons across phylogenetic lines can revealessential structural features of both RNAs and proteins. Thistechnique has been beautifully exploited in establishing con-served loops and helices in 16S and 23S rRNAs (see, e.g.,reference 236) but has been less well developed to date inanalysis of ribosomal proteins. Golden et al. (213) haverecently reported the three-dimensional structure of ribosomalprotein S17 from B. stearothermophilus, based on nuclearmagnetic resonance spectroscopy, and Hoffman et al. (266)have solved the crystal structure for protein L9 from thisbacterium. The comparative analysis presented in both thesepapers includes the sequences for the homologous proteinsfrom pea and Arabidopsis thaliana, as well as the CyanophoraS17 and Synechocystis L9 sequences. Conserved structuralresidues and proposed rRNA-binding sites can be identified inboth proteins. Conservation of the length of an al-helix in theL9 protein, for example, suggests that this helix has a structuralrole, whereas variability in the central region of the proteinsequence is consistent with its occupying an exposed position in

the ribosome. Similar analyses should be possible with otherribosomal proteins.

ASSEMBLY OF CHLOROPLAST RIBOSOMES

Subramanian (606) points out that the ribosomal proteinsencoded in land plant chloroplast genomes share the followingproperties: (i) all are important proteins in early steps inribosome assembly as judged from comparison with assemblymaps of the E. coli 30S and 50S subunits (see, e.g., reference707); (ii) their loss is likely to be lethal, since no E. coli mutantslacking any of these proteins, with the exception of L33, havebeen isolated; and (iii) all are basic or highly basic ribosomalproteins, even though chloroplast ribosomes contain a muchlarger number of acidic ribosomal proteins than E. coli ribo-somes do.The limited data available on chloroplast ribosome assembly

have been summarized for land plants by Mache (377). Themain observations on land plants and C. reinhardtii are asfollows. (i) Seven chloroplast ribosomal proteins, four of whichare made in the chloroplast, bind to chloroplast or E. coli 16SrRNA, in agreement with the seven E. coli ribosomal proteinsknown to bind to 16S rRNA (531). (ii) Two 5S rRNA-bindingproteins have been detected in spinach (L22 and "CS-12"[651]), in contrast to three in E. coli (L5, L18, L25). However,one of these chloroplast proteins, encoded by the rp122 gene,has a central region of homology to other L22 proteins flankedby long N- and C-terminal extensions (76). (iii) In C. rein-hardtii, the second step of processing of "L-18," a homolog ofE. coli L27, occurs during ribosome assembly and may berequired for maturation of the 50S ribosome subunit (363). (iv)The nucleus-encoded ribosomal protein "L-29" of C. rein-hardtii is required for assembly of chloroplast-encoded ribo-somal protein "L-13" (see below and reference 446).Mutants with defects in chloroplast ribosome assembly have

been identified in C reinhardtii and map to seven nuclear andtwo chloroplast loci (see reference 247 for a summary). Twophenotypic classes are seen, one in which small subunits aredeficient but large subunits accumulate and one in which bothsubunits are deficient. No mutant specifically deficient in largesubunits has been identified. Analysis of double-mutant com-binations of five nonallelic nuclear mutations led to theproposal that mutants deficient in both subunits were blockedin steps common to the assembly of the two subunits, while themutants that accumulated large subunits were blocked only inthe assembly of small subunits (248). One of the mutantsdeficient in both subunits, ac-20, has subsequently been shownto be defective in its ability to splice an intron present in theprecursor of 23S rRNA (259). Since expression of the mutantdefect apparently occurs after processing of the primary rRNAtranscript, these observations suggest that inability to processpre-23S rRNA properly results in a deficiency of large sub-units, which in turn prevents small subunit assembly.Two Chlamydomonas allelic nuclear mutations, cr-6 and

cr-7, cause production of ribosomal large subunits that sedi-ment abnormally on sucrose gradients, assemble into mono-mers less efficiently than those from wild-type cells, and showreduced capacity for protein synthesis in vivo (446). Largesubunits of chloroplast ribosomes from these two mutants lacktwo proteins, one of which ("L-29") is made in the cytoplasmand the other ("L-13") is made in the chloroplast. The primarydefect appears to be an inability to make "L-29," whichprevents assembly of "L-13" into the 50S subunit. Immunolog-ically, "L-29" is related to E. coli L23 and to a lesser extent toL7/L12, while "L-13" is related to E. coli L5 (see above andreference 510). Assembly of L5 into the E. coli 50S subunit

MICROBIOL. REV.


does not depend directly on the presence of either L7/L12 orL23 (707).

Chloroplast and nuclear mutations causing complete ornearly complete loss of chloroplast ribosomes from whitetissues and seedlings have also been identified in land plants(39) and have been very useful in probing the function of thechloroplast protein-synthesizing system. Barkan (17) has re-cently described transposon-induced nuclear mutations inmaize that impair chloroplast protein synthesis. Seedlings ofthese mutants are paler green than those of the wild type andare photosynthetically inactive, although they do accumulatenucleus-encoded proteins of the light-harvesting complex. Onemutant appears to be blocked specifically in processing of 16SrRNA.Complete assembly of ribosomal subunits has been detected

in isolated spinach chloroplasts, implying either that a pool ofunassembled nucleus-encoded ribosomal proteins exists inplastids or that ribosomal proteins can be released frompreexisting ribosomes and reutilized (121). Certain proteins,e.g., the CS-S5 protein of spinach, can be found in highconcentrations in the chloroplast stroma (741). When ryeplants are grown at high temperature (32°C), plastid ribosomeformation is severely impaired (168, 169). Pools of a fewunassembled plastid ribosomal proteins were detected whensoluble extracts from leaves deficient in 70S ribosomes wereexamined with antibodies raised against purified50S and 30Ssubunits. These antibodies were shown to react with about 17of the 33 polypeptides of the 50S subunit and 10 of the 25proteins of the 30S subunit. Feierabend and Berberich (168)believe that these observations confirm the absence of plastidribosomes following bleaching and that the unassembled pro-teins detected by the antibodies are probably of cytoplasmicorigin.

SYNTHESIS OF THE COMPONENTS OFCHLOROPLAST RIBOSOMES

Biogenesis of chloroplast ribosomes requires expression ofboth nuclear and chloroplast genes encoding different ribo-somal proteins, as well as chloroplast genes encoding thecomponent rRNAs. The mechanisms by which the appropriatestoichiometry of these components is achieved from genespresent in vastly different copy numbers remain poorly under-stood (229, 377, 379). In general, nuclear gene expression inplants tends to be controlled at the level of transcription and tobe subject to light regulation, whereas chloroplast gene expres-sion is largely regulated posttranscriptionally. Pool sizes ofsome components may also be controlled by proteolysis.Furthermore, chloroplast genes encoding ribosomal compo-nents appear to be regulated differently from those encodingproteins of the photosynthetic apparatus (207, 253, 365). Inthis section we will discuss transcription and splicing of rRNAs,transcription and translation of genes encoding ribosomalproteins, and posttranscriptional and translational regulationof chloroplast gene expression.

Transcription of rRNA Genes

As in bacteria, chloroplast rRNA genes are thought to betranscribed as large precursor molecules that subsequentlyundergo several processing steps to generate the maturerRNAs (114). Relatively little is known, however, about thespecific enzymes and cleavage steps that are involved. Al-though 5S rRNA sequences are not detected in the primarytranscript, S1 and primer extension experiments suggest thatthe 5S gene is indeed cotranscribed with the 16S and 23S genes

and that the 5S rRNA is rapidly cleaved from the initialprecursor (10, 601). Possible promoter sequences have beenfound between the 4.5S and 5S rRNA genes in some plants,suggesting that separate 5S transcription might occur, butthese sequences are not present in many plants and do notseem to be active in vitro (10, 146). Likewise, the tRNA'gfollowing the 5S gene in land plants is thought to be cotrans-cribed with the rRNAs (118). However, the tRNAVal upstreamof the 16S gene in many plants lies distal to the identifiedtranscription start sites for the rRNA operon and thus is notpart of the primary transcript.

Possible processing sites of the primary rRNA transcripthave been identified by Si and reverse transcriptase mapping(118, 601; see also reference 114). Experiments so far suggestthat cleavage at the various sites does not occur in a preciseorder. The 5'- and3'-terminal precursor sequences of the 16Sand 23S RNAs can form double-stranded stem structuressimilar to those of the E. coli rRNA genes, leading to thesuggestion that these molecules may be processed by an RNaseIII-like endonucleolytic cleavage (114). Vera et al. (669) havefound that tobacco chloroplast ribosomes contain a minorfraction of 16S rRNA molecules in which a 30-nt leadersequence containing the putative RNase III site is still present,suggesting that the final maturation of the 16S rRNA mayactually take place within the ribosome.

Processing of the tRNAs of the spacer between the 16S and23S genes requires the ribozyme RNase P (679), but otherprocessing reactions specific to plant chloroplast rRNAs havenot been well characterized to date. Additional processing ofthe mature 23S rRNA of land plants may occur, leading tohidden breaks at specific stem-loop sites. Thus 23S rRNAisolated under denaturing conditions typically appears as sev-eral short species rather than a single intact molecule. Delpand Kossel (114) suggest that this fragmentation is real, not anartifact of preparation, and that it may be necessary for somestructural or functional requirement of the chloroplast ribo-some.There are conflicting data regarding whether chloroplast

rRNA genes are transcribed by a different RNA polymerasefrom that used to produce mRNAs (229, 283, 320, 349, 642).Chloroplast genomes typically contain genes homologous tothe rpoA, rpoB, and rpoC genes, which encode RNA poly-merase subunits of bacteria, but appear to lack a gene for rpoD,which encodes the principal cr factor of the bacterial RNApolymerase (24, 171, 614, 653). However, immunological stud-ies suggest that the chloroplast RNA polymerase complexes docontain a-like factors (642, 653). Expression of the chloroplast-encoded RNA polymerase genes in Euglena chloroplasts re-sulted in activity of a soluble RNA polymerase fraction capableof transcribing tRNA and mRNA genes, whereas transcriptionof rRNA genes required a membrane-bound fraction (226,244, 361). However, soluble RNA polymerase from spinachchloroplasts appears to be able to transcribe both rRNA andprotein-coding genes in vitro (55). Fractionation of RNApolymerase activities from spinach suggests that a 110-kDacomponent may represent a core enzyme active as a singlepolypeptide chain, which shows no immunological similarity toE. coli RNA polymerase subunits and is thus probably not theproduct of the chloroplast rpoB gene (349). Further evidencefor a second, presumably nucleus-encoded RNA polymeraseactivity in chloroplasts comes from the observation that theplastid genome of the parasitic plant Epifagus virginiana lacksthe rpo genes but is nevertheless transcribed (115, 435, 713,714). Also, some plastid genes in heat-bleached leaves of ryeand barley plants and the albostiians mutant of barley aretranscribed, although these leaves lack functional chloroplast

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732 HARRIS ET AL.

ribosomes and are thus unable to translate the mRNAs for theplastid-encoded rpo genes (164, 262, 263).

In land plant chloroplasts, promoter sequences precedingthe transcription start sites of rRNA operons do not differsignificantly from the -10 and -35 consensus sequences ofplastid protein-coding genes and are typically 50 to 200 bpupstream of the 16S rRNA genes (229). The resemblance topromoter sequences of protein-coding genes implies that a

single-core RNA polymerase might be able to transcribe allclasses of RNAs, a notion that is also supported by thedemonstration that a chimeric gene consisting of the 16Spromoter fused to the bacterial aadA gene encoding spectino-mycin and streptomycin resistance is expressed in chloroplasttransformants of tobacco (621). This construct was edited bysite-directed mutagenesis to eliminate upstream AUGs in themRNA, and a synthetic leader sequence containing a ribo-some-binding site was attached. The aadA gene was followedby the 3' region of the chloroplast psbA gene. However,deletion analysis with chloroplast transformants in which pu-tative promoter regions were fused to a reporter gene has ledto the identification of two classes of chloroplast promoters inC. reinhardtii (310). Promoters of the first class, such as atpB,lack a conserved -35 sequence, and deletion of this region hasno effect on relative rates of transcription or the transcriptioninitiation site. The second class of promoters includes the 16SrRNA gene and has a conserved and essential -35 sequence.A 14-bp sequence that is recognized by polypeptides of 33 and35 kDa has also been identified upstream of the 16S rRNAinitiation start site in spinach (13). This sequence is not foundupstream of chloroplast genes encoding mRNAs or tRNAsand thus may have a role in differential regulation of rRNAand protein-coding genes during chloroplast development.

Expression of rRNA genes appears to depend on both lightand developmental stage in plant seedlings (309, 442), butsteady-state levels of rRNA seem to be controlled more by therate of breakdown than by transcriptional regulation (114).Bendich (21) has suggested that rRNA transcription is regu-lated primarily by gene dosage. However, in cells of C.reinhardtii grown in the presence of 5-fluorodeoxyuridine, thereduction in chloroplast DNA copies was mirrored by areduction in accumulation of chloroplast rRNA (208, 272).There is indirect evidence that conserved stem-loop structuresand short open reading frames found between the promoterand the start of the 16S coding sequence could be involved inregulation of rRNA operon expression in spinach chloroplasts(338).

Bisanz-Seyer et al. (27) observed the accumulation of 16SrRNA and mRNAs for several chloroplast ribosomal proteinsduring early development of spinach. The 16S rRNA, alreadypresent in dry seeds, began to increase at the time of seedgermination 5 days after planting and continued to accumulatethereafter. Most ribosomal protein mRNAs appeared at thebeginning of germination (5 days), but the rpsl9 and rp123mRNAs appeared 2 days earlier. These two genes belong to alarge chloroplast ribosomal protein operon including parts ofthe S10, spc, and a operons of E. coli. Interestingly, mRNAsfor several other ribosomal protein genes in this operon,including rps3 and rp116, did not begin to accumulate untilgermination. One explanation of these results is that the firstthree genes in this operon, rp123-rp12-rpsl9, are transcribedearly and the whole operon is transcribed later. This hypothesispredicts that rp12 transcripts should also be detected early, butthis was not examined with appropriate probes. Alternatively,the entire operon may be transcribed early, but transcriptsdistal to rpsl9 are initially degraded.

Gantt et al. (194) have presented additional evidence that

nuclear genes encoding chloroplast ribosomal proteins aresubject to light regulation. Pea seedlings grown in bright lightin the presence of the inhibitor norflurazon, which blockscarotenoid synthesis, showed greatly decreased levels ofmRNA for nucleus-encoded ribosomal proteins comparedwith seedlings grown with or without norflurazon in the dark.Levels of mRNAs for other chloroplast components weresimilarly diminished, but mRNAs for cytoplasmic ribosomalproteins, histones, and other nonphotosynthetic proteins werenot affected.

Transcription of Chloroplast Genes EncodingRibosomal Proteins

Nuclear and plastid genes which cooperate in controllingchloroplast biogenesis and function appear to be regulated byvery different mechanisms, although their gene products oftenoccur in equal stoichiometry within the multimeric thylakoidcomplexes or chloroplast ribosomes. This may reflect the wayin which plant cells cope with large differences in ploidy levelsbetween nuclear genes (present in single copies or small genefamilies) and chloroplast genes (present in hundreds or thou-sands of copies per cell). Nuclear genes encoding chloroplastpolypeptides are regulated largely at the transcriptional level inresponse to environmental and developmental signals (forreviews, see references 209, 329, and 443). In contrast, mostplastid genes appear to be transcribed at all times duringplastid development, and posttranscriptional regulatory mech-anisms are thought to play major roles in modulating theirexpression (see below).

All 21 of the ribosomal protein genes in the rice, tobacco,maize, and Marchantia chloroplast genomes have been dem-onstrated to be transcribed (540), but not all have been shownunequivocally to be translated into the corresponding polypep-tides. N-terminal sequencing indicates that ribosomal proteinsS12, S16, S19, L2, L20, L32, L33, and L36 of spinach chloro-plasts do indeed appear to be products of the correspondingchloroplast genes (540). Most of the clusters of chloroplast-encoded ribosomal protein genes that show striking homologyto bacterial ribosomal protein operons (631) (Fig. 5) areprobably functional transcriptional units (540). Multiple tran-scripts are typically found for individual ribosomal proteingenes in these clusters, and these may arise from processing oflarger polycistronic transcripts (470, 605). In C. reinhardtii, nosingle large mRNA has been detected for the ribosomalprotein gene cluster that begins with the rp123 gene, but,rather, a series of transcripts of different lengths have beenobserved, including some probably monocistronic transcriptsand others corresponding to two or more genes (362; see alsoreference 253).

Christopher and Hallick (87, 88) published a detailed char-acterization of the organization and transcription of the largeribosomal protein operon in Euglena gracilis. The primarytranscript of this operon includes 11 genes encoding ribosomalproteins, a tRNA gene, and an open reading frame encoding ahighly basic protein of unknown function (88, 243). An 8.3-kbmRNA from which all introns have been removed by splicingis then processed stepwise into transcripts containing one ormore genes.Tonkyn and Gruissem (648) examined the relative expres-

sion levels of the intact S10 operon from spinach and thepartial S10 operon that begins in the opposite inverted repeatbut ends in an rpsl9' pseudogene as discussed above. Becausethe upstream regulatory regions of these two operons areincluded in the inverted repeat in spinach and are thereforeidentical, Tonkyn and Gruissem predicted that these operons

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might be expressed at the same level and that a nonfunctionalproduct of the rpsl9' gene might accumulate. In fact, theyfound that the rpsl9' transcript was present at very low levelsif at all and that the rp12 mRNA that is translated appears to betranscribed from the gene copy located in the completeoperon. However, transcription of the intact operon can beinitiated from several different promoters, suggesting that itmay be subject to developmental regulation.A number of genes encoding ribosomal proteins are part of

mixed clusters that also contain genes encoding components ofthe photosynthetic apparatus. Stahl et al. (587) demonstratedthat such a mixed chloroplast gene cluster in maize containinggenes encoding four subunits of the ATP synthase (atpI, atpH,atpF, and atpA) and the gene encoding ribosomal proteinS2(rps2) produces a total of 12 transcripts, including a majorspecies of 6,200 nt containing mRNAs of all five genes. Theysuggest that this plastid gene cluster is "functionally organizedas an operon with additional regulatory features to allow forincreased accumulation of mRNAs for the thylakoid compo-nents."The analogous operon in Euglena gracilis, containing rps2,

atpI, atpH, atpF, atpA, and rpsl8, was analyzed by Drager andHallick (134). Of these genes, all but atpH contain one or moreintrons, comprising in aggregate nine introns of the group IIIclass unique to the genus Euglena and its colorless relativeAstasia (87, 565), seven introns with group II structure, andone intron that matched neither category. Drager and Hallick(134) found that all 17 introns are removed to yield a 5.5-kbmRNA spanning all six genes, from which monocistronictranscripts are then generated, presumably by endonucleolyticcleavage. The unique 434-nt intron in the rpsl8 gene is acomplex twintron, consisting of four group III introns whichare removed in four sequential splicing reactions, some ofwhich can use multiple splice sites (135).Chen et al. (83) found that the psaA, psaB, and rpsl4 genes

in rice are organized into a single transcriptional unit. A 5.2-kbtranscript hybridizing to probes for all three genes was ob-served in leaf tissue. Ribosomal protein L32 of the tobaccochloroplast has been shown to be encoded by the geneformerly identified as open reading frame ORF55 (733),located in the small single-copy region. A primary transcript of1,550 nt contains no other open reading frames and overlapsthe ndhF gene on the opposite strand (668). Vera et al. (668)demonstrated that the rp132 promoter is located within thendhF coding region, the first instance so far of an internaldivergent promoter in the chloroplast genome.

Posttranscriptional Regulatory MechanismsAffecting Chloroplast mRNAs

Recent reviews by Rochaix (520), Gruissem and Schuster(228), and Gruissem and Tonkyn (229) provide excellentsummaries of the current literature dealing with the process-ing, stability, and translational control of chloroplast mRNAsin general, although relatively little of this literature is specificto genes encoding ribosomal proteins. Two generalizationsemerge from these reviews. First, nuclear gene products con-trol expression of chloroplast-encoded mRNAs. On the basisof analysis of nuclear nonphotosynthetic mutants of C. rein-hardtii, several different gene products may be required forexpression of a given chloroplast mRNA (520). Second, spe-cific proteins bind to inverted repeat regions present in the 5'and 3' untranslated leaders of chloroplast mRNAs that arecapable of forming thermodynamically stable stem-loop struc-tures (105, 229). Although the 3' untranslated leaders appearto function in stabilization and processing of mRNAs rather

than as transcript terminators in land plants, there is someevidence that they serve as terminators for chloroplast tran-scripts in C. reinhardtii (30). Experiments are currently underway in several laboratories to demonstrate the functional roleof individual binding proteins in mRNA stability and transla-tion as well as to decipher the signal transduction pathwayleading to the expression of these proteins. For example,Nickelsen and Link (456) have described a 54-kDa proteinfrom mustard chloroplasts that binds to a conserved sequencein the 3'-flanking regions of the tmK and rpsl6 genes andappears to have endonucleolytic activity that may be involvedin RNA3'-end formation and mRNA stability.Development of reliable protocols for chloroplast transfor-

mation in C. reinhardtii (48) and tobacco (390), coupled withthe ability to express foreign reporter sequences in the chlo-roplast, has allowed the functional dissection of the 5' and 3'untranslated leaders for the first time (84, 214, 535, 591, 621).Detailed consideration of the genetic basis for translationalregulation in chloroplasts and mitochondria and a modeldepicting the role of a multiprotein translation complex boundto the 5' untranslated leaders of organelle mRNAs in modu-lating the translational regulation are presented in a recentreview (207).

Membrane Binding of Chloroplast Ribosomes

Thylakoid-bound polysomes have been characterized inchloroplasts with respect to their physical status and physio-logical function (see references 40, 41, and 286 for reviews).High-salt washes of isolated thykakoids remove 30 to 45% ofthe membrane-bound RNA while addition of puromycin re-leases up to 80% of the bound RNA. By analogy with therough endoplasmic reticulum, these results suggest that be-tween one-third and one-half of the polysomes found onthylakoids are attached electrostatically to the membranes andthe rest are held by both electrostatic forces and nascentpolypeptide chains. The electrostatic binding of chloroplastpolysomes predicts the presence of a ribosome receptor similarto the ribophorin-containing receptor found on the roughendoplasmic reticulum (503) but missing from the bacterialcytoplasmic membrane. Other components of the system forsynthesizing eukaryotic secretory proteins, such as a signalrecognition particle and a docking protein, have not beendemonstrated in chloroplasts. The fraction of membrane-bound polysomes observed can be markedly enhanced bypretreating the cells with antibiotics such as chloramphenicoland erythromycin that inhibit transpeptidation.Freimann and Hachtel (180) examined the distribution of

mRNAs on free and membrane-bound chloroplast polysomesof broad bean (Vicia faba). They used the criteria of release ofthe associated mRNA by high salt alone or high salt pluspuromycin, together with gene-specific probes, to distinguishmRNAs electrostatically bound to thylakoids from those en-gaged in cotranslational protein synthesis. Three classes ofmRNA were recognized. (i) The rbcL mRNA encoding thelarge subunit of Rubisco was the only mRNA associated solelywith stromal polysomes. However, other authors have reportedthat rbcL mRNA is also found associated with thylakoidmembranes (252). (ii) Thylakoid polysomes containingmRNAs for six genes encoding integral membrane proteinsappeared to synthesize their products in a cotranslationalfashion. These mRNAs were released only by high salt pluspuromycin. (iii) Thylakoid polypeptides encoded by sevenother genes were assumed to be incorporated posttranslation-ally because their mRNAs were found on stromal polysomes orpolysomes bound electrostatically to the thylakoid membranes.

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Other supporting evidence exists that such chloroplast-synthe-sized proteins as Dl, the reaction center polypeptide encodedby the psbA gene, and the alpha and beta subunits of the CF1portion of the ATP synthase are made, at least in part, onthylakoid-bound polysomes (see reference 286 for a summary).Membrane binding of polysomes may play an additional role

in translational regulation of chloroplast gene expression. In C.reinhardtii, the distribution of chloroplast mRNAs variedbetween the thylakoid and soluble fractions in cells growingsynchronously on a light-dark cycle (41). Thus, a strikingincrease in the fraction of membrane-bound polysomes wasobserved for both rbcL and psbA mRNAs in the light period.Thylakoid binding may occur in the light phase becausetranslation is initiated. In contrast, Klein et al. (308) found thatthe psaA, psaB, and psbA transcripts are primarily membraneassociated in dark-grown barley plants. The protein productsof these genes are made in the dark but are unstable in theabsence of chlorophyll (441). Jagendorf and Michaels (286)correctly point out that the possible role of the thylakoidmembrane itself in translational regulation requires furtherinvestigation.

HOW ESSENTUIL IS CHLOROPLASTPROTEIN SYNTHESIS?

Chloroplast protein synthesis has long been known to beindispensable for survival of plants and algae that depend onCO2 as their sole carbon source, since numerous proteinsrequired for photosynthesis are plastid gene products. How-ever, as we learn more about the genes encoded in the plastidgenomes of algae such as Cyanophora, Cryptomonas, andPorphyra species, some of which specify proteins required foramino acid or fatty acid biosynthesis, the likelihood is increas-ing that chloroplast protein synthesis is also required for theproduction of one or more essential proteins not involved inphotosynthesis. This viewpoint is supported by some, but notall, analyses of plastid genome function in colorless plants. Webegin with cases that support the hypothesis that chloroplastprotein synthesis is essential and then turn to evidence thatmakes the converse argument.The colorless heterotroph Astasia longa is closely related to

Euglena gracilis and possesses a circular 73-kb plastid genome.This genome is the counterpart of the larger (145-kb) Euglenachloroplast genome, and the genes identified include therRNAs, tufA, and several tRNAs and ribosomal proteins(565-569). The rbcL gene encoding the large subunit of theenzyme Rubisco is the only photosynthetic gene so far detectedin the Astasia plastid genome. This polypeptide has beenimmunoprecipitated from Astasia longa, suggesting that therbcL gene is transcribed and translated and that the plastidprotein-synthesizing system of this colorless flagellate must befunctional. Colorless, heterotrophic algae of the genus Poly-toma, closely related to or derived from the genus Chlamydo-monas, contain a plastid genome (ca. 200 kb) similar in size tothe C. reinhardtii chloroplast genome (574, 670). Plastid rRNAgenes are present and expressed in Polytoma species, leuko-plast ribosomes have been isolated, and the tufA gene has beenidentified. These results suggest that Polytoma species too havea functioning plastid protein-synthesizing system (575, 576,670).

Plastid genomes of the colorless plants Epifagus virginiana(beechdrops) and Conopholis americana in the Orobran-chaceae family of root-parasitic angiosperms have also beenexamined (115, 702-704, 711, 712, 714). The 70-kb Epifagusplastid genome has been completely sequenced and containsonly 42 genes (714). At least 38 of these genes encode

components of the plastid gene expression system (rRNAs,tRNAs, and ribosomal proteins). Functional photosynthesisgenes and genes of the NADH dehydrogenase complex areabsent, although several photosynthetic pseudogenes havebeen found. The Epifagus plastid genome contains only 17tRNAs, suggesting that tRNAs must be imported if this plastidprotein-synthesizing system is to function. Genes specifying thefour RNA polymerase subunits encoded in the chloroplastgenomes of green plants are also absent. Although no exper-iments with Epifagus virginiana demonstrating synthesis of aspecific plastid-encoded protein have been reported, there areseveral lines of indirect evidence suggesting that the plastidprotein-synthesizing system is functional. Wolfe et al. (714)reported transcription of all eight rRNA and protein-encodinggenes so far examined and cited the following three evolution-ary arguments in favor of function. (i) Plastid gene deletions inEpifagus virginiana are not random but are skewed towardphotosynthetic genes. Although only 5% of photosyntheticsequences have been retained with respect to tobacco, 80% ofthe ribosomal protein sequences are present. (ii) Large openreading frames are retained in the Epifagus plastid genome. Ifthese genes were nonfunctional, mutations, truncations, andinternal deletions would have been expected to occur, as is trueof pseudogenes in the Epifagus plastid genome. (iii) Thegenera Conopholis and Epifagus share the loss of the photo-synthetic and ndh genes, but their rRNA genes are stronglyconserved, suggesting that the evolution of these genes isconstrained by natural selection because they are functional.Why might the protein-synthesizing systems of Epifagus

virginiana and other colorless plants be essential? The argu-ment of Howe and Smith (275) that plastid protein synthesiswas retained in Epifagus species for the sole purpose of makingthe chloroplast-encoded RNA polymerase subunits requiredfor transcription of the tRNAGIU gene necessary for porphyrinsynthesis (see, e.g., references 20 and 552) is invalidated by thefinding that the RNA polymerase subunit genes are absentfrom the Epifagus plastid genome (714). However, one or moreother proteins essential for survival might be encoded andtranslated in the Epifagus chloroplast. The best candidate isclpP, which specifies one subunit of the plastid homolog of theATP-dependent Clp protease of E. coli. Perhaps this proteaseis involved in processing chloroplast protein precursors into anactive form or in protein degradation.The thesis that chloroplast protein synthesis is essential is

also supported by work on the genus Plasmodium, the malariaparasite. This protozoan contains, in addition to its tiny (6-kb)linearly reiterated mitochondrial genome, a 35-kb circularDNA molecule that seems to be a residual plastid genome (seereferences 273, 484, 700, and 701 for reviews). The 35-kb circlepossesses inverted repeats containing continuous rRNAs withsecondary structures quite similar to those predicted for E. coli(166, 197, 198). It also encodes tRNAs, two subunits of aeubacterial-type RNA polymerase, and at least four ribosomalproteins (165).

Genetic evidence suggests that chloroplast protein synthesisin C. reinhardtii is essential for survival. Hanson and Bogorad(245) showed that cells carrying a nuclear mutation conferringerythromycin resistance on chloroplast ribosomes underwent amarked reduction in chloroplast ribosome content whenshifted from 25 to 15°C. Ribosome loss was accompanied byloss of the ability of the mutant to grow at 15°C under allconditions. Also, although several Chlamydomonas mutantswhich have a reduced content of chloroplast ribosomes havebeen isolated, none is completely deficient in chloroplastprotein synthesis (247-249, 446). Lastly, mutations with sym-metric deletions of the psbA gene encoded within the inverted

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repeat are frequently isolated, but the only known deletionmutation affecting the rRNA gene region of the repeat re-moved only one set of rRNA genes (486). Many years ago,Blamire et al. (29) reported that treatment of wild-type cellswith antibiotics blocking translation on chloroplast ribosomesinhibited replication of nuclear but not chloroplast DNA.Inhibition did not occur in mutant strains with chloroplastribosomes resistant to these antibiotics. These intriguing ex-periments have never been repeated.The notion that chloroplast protein synthesis is indispens-

able is challenged by several other findings. Cuscuta reflexa(Convolvulaceae) is a colorless parasitic plant, unrelated to thegenus Epifagus, that contains residual thylakoids and traces ofchlorophylls a and b (376). The plant also possesses very lowlevels of light-stimulated CO2 fixation and Rubisco activity,although the Rubisco large subunit is undetectable by immu-nological methods (237, 376). Partial sequence analysis of theCuscuta reflewa plastid genome (33, 237, 238) revealed thatalthough many photosynthesis genes are intact (e.g., atpB,atpE, rbcL, and psbA), a large deletion has removed certainprotein synthesis genes (rpl2, rp123, and several tRNA genes),and the ndh genes appear to be nonfunctional. Transcriptionanalysis showed that rbcL was weakly transcribed while psbAwas transcribed strongly. Bommer et al. (33) hypothesized thatthe translational apparatus of Cuscuta reflexa is nonfunctionalbased on the absence of specific protein synthesis genes fromthe plastid genome and their inability to detect Rubisco largesubunit using immunological techniques. Alternatively, theplastid protein-synthesizing system of Cuscuta reflexa might befunctional, with the missing tRNAs and ribosomal proteinsencoded in the nucleus.

In tobacco, antibacterial antibiotics such as streptomycinand lincomycin cause bleaching, but they do not cause death ofcallus in tissue culture (389). The bleached antibiotic-sensitivecells continue to divide at a reduced rate, using sucrose as thecarbon source. Mutants resistant to these antibiotics are greenin tissue culture. Streptomycin-resistant mutants result fromspecific base pair changes in the 16S rRNA or rpsl2 genes,while those resistant to lincomycin arise because of a specificbase pair alteration in the gene encoding the 23S rRNA (391,590) (Table 2). The ability of bleached antibiotic-sensitive cellsto continue to divide implies that chloroplast protein synthesismay be required only for the manufacture of photosyntheticand ribosomal proteins in Nicotiana species. However, tobaccocalli containing a chloroplast mutation to streptomycin resis-tance grow better in the dark on antibiotic than do sensitivecalli (98, 392).

Experiments with calli cultured from roots of haploid riceplants derived from pollen grains provide convincing evidencethat plastid protein synthesis is not essential in this system(246). Albino plants obtained in this way from barley andwheat have long been known to contain large deletions in theplastid genome (106, 107), but the deleted molecules form aheterogeneous collection. By inducing callus cultures fromroots of albino rice plants, Harada et al. (246) obtained isolatesthat were homoplasmic for different large deletions. Of fivethat were characterized, four lacked the inverted repeat andthe plastid rRNA genes, so that none of these callus culturescould carry out chloroplast protein synthesis, yet all fiveretained one region in common which contained the tRNAGlugene. Harada et al. (246) suggest that this gene has beenretained because the tRNAGlu encoded by the gene is essentialfor porphyrin biosynthesis. Similarly, heat-bleached leaves ofrye and oat lack chloroplast ribosomes but have substantialamounts of tRNAGlU and chlorophyll synthetase activity de-spite their low chlorophyll level (262). Obviously, expression of

the tRNAGlU gene would require functioning of a nucleus-encoded plastid RNA polymerase. Retention of small amountsof plastid DNA in bleached Euglena mutants (254) is probablynot related to a general requirement for tRNAG'U in porphyrinsynthesis, since mitochondrial heme in this flagellate is madevia the animal-type 8-aminolevulinic synthetase pathway whichdoes not require tRNAGlU (268). In fact, the rRNA genes werethe only ones detected in these deleted plastid genomes.The existing data currently suggest that chloroplast protein

synthesis may be essential for survival in Chlamydomonas andEpifagus species but possibly not in other plants such astobacco, at least in tissue culture. The plastid-encoded tRNA-Glu gene is essential for synthesis of all porphyrins in plants andalgae examined to date, with the exception of Euglena gracilis,so transcription of this gene is crucial to survival. However, theEpifagus results suggest that in this plant, at least, transcriptionof tRNAGlU depends on a nucleus-encoded RNA polymerase.

CONCLUSIONS

Analysis of chloroplast sequences has been invaluable indetermining variable and conserved regions of the 16S and 23SrRNA molecules and in predicting their secondary and tertiarystructures. Similar comparisons of ribosomal protein se-quences are just beginning but will doubtless prove importantin years to come. Specific domains conserved over a widevariety of organisms are likely to be important in ribosomefunction or assembly. Sequence analysis of ribosomal proteinsin a diverse array of algae and land plants will allow furtherrefinements in understanding which domains are important forribosome function. Although no analysis of mitochondrialribosomal proteins has been included here, these will also be avaluable comparative tool in such research. We hope that thisreview will provide a useful starting point for investigations onthese topics.

ACKNOWLEDGMENTS

We thank Hans Bohnert, Donald Bryant, Robin Gutell, ClaudeLemieux, Xiang-Qin Liu, Michael Reith, Alap Subramanian, andMonique Turmel for sharing unpublished data.Our work described in this review was supported by NIH grant

GM-19427.

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