Plant Molecular Biology 11:293-299 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands 293

The nucleotide sequences of barley cytoplasmic glutamate transfer RNAs and structural features essential for formation of ~i-aminolevulinic acid

David Peterson, Astrid Sch6n and Dieter $611" Department of Molecular Biophysics & Biochemistry, Yale University, P.O. Box 6666, New Haven, CT 06511, USA (*author for correspondence)

Received 14 March 1988; accepted 23 May 1988

Key words: tRNA °u, glutamyl-tRNA synthetase, glutamyl-tRNA reductase, substrate specificity

Abstract

In chloroplasts and a number of prokaryotes, &aminolevulinic acid (ALA), the universal precursor of porphy- rins, is synthesized by a multistep enzymatic pathway with glutamyl-tRNA °u as an intermediate. The ALA synthesizing system from barley chloroplasts is highly specific in its tRNA requirement for chloroplast tRNAGIU; a number of other Glu-tRNAs are inactive in ALA formation although they can be glutamylated by chloroplast aminoacyl-tRNA synthetases. In order to obtain more information about the structural features defining the ability of a tRNA to be recognized by the ALA synthesizing enzymes, we purified and sequenced two cytoplasmic tRNA °u species from barley embryos which are inactive in ALA synthesis. By using glutamylated tRNAs as a substrate for the overall reaction, we showed that GIu-tRNA reductase is the enzyme responsible for tRNA discrimination.

Introduction

The first committed precursor in the pathway of por- phyrin synthesis in all organisms investigated to date is 6-aminolevulinic acid. This compound is synthe- sized from succinyl-coenzyme A and glycine in non- photosynthetic eukaryotes, most bacteria, and algal mitochondria (for a review, see [1]). In contrast to this well characterized one-step biosynthesis, ALA is derived from glutamate in a multi-step pathway found in chloroplasts, photosynthetic prokaryotes and some anaerobic bacteria [2, 4, 13, 15, 16, 26].

Surprisingly, the conversion of glutamate to ALA in barley chloroplasts requires chloroplast tRNA 61u as a cofactor [8, 22]. Studies have shown that gluta- mate is first activated by ligation to chloroplast tRNA Glu by the chloroplast glutamyl-tRNA synthe- tase (GIuRS) [6, 22]. In a second step the C- 1 activat-

ed glutamate serves as a substrate for an NADPH- dependent GIu-tRNA reductase (also called "de- hydrogenase" in the literature) which converts it to glutamate- 1-semialdehyde (GSA) [12]. This com- pound, in turn, is transformed to tS-aminolevulinic acid by a transaminase [9, 26].

The overall reaction of ALA synthesis in barley chloroplasts is highly specific for the cognate chlo- roplast tRNAGlu; neither chloroplast tRNA GIn, nor E. coli tRNA Glu, yeast, or wheat germ tRNAs can substitute for the homologous RNA [8]. Since barley chloroplast GluRS aminoacylates a number of tRNAs that are not active in the overall reaction [21], the GIu-tRNA reductase must be able to dis- criminate between different glutamate accepting tRNAs. In this paper we report the purification and sequence analysis of two cytoplasmic barley gluta- mate tRNA species and some results concerning the

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t R N A specificity o f barley chloroplast G l u - t R N A reductase.

M a t e r i a l s a n d m e t h o d s

General

Barley seed (Hordeum vulgare var. "Sval6fs Bo-

nus" ) was obtained f rom the Carlsberg Laboratory. Plaskon C T F E 2300 powder was obtained f rom A1- lied Chemical Corp. Adogen 464 ( tr ialkylmethylam- moniumchlor ide) was f rom Ashland Chemical Co. D E A E cellulose (type 52) was f rom W h a t m a n Bi- osystems, Ltd. Cellulose thin-layer plates were pur- chased f rom Merck and PEI-cel lulose thin-layer plates were purchased f rom Macherey-Nagel .

Nucleases T1, T2, and U 2 w e r e obtained f rom Calbi- ochem. RNAse A and alkaline phosphatase (calf in- testine) were purchased f rom Boehringer Mannhe im Biochemicals. Staphylococcus nuclease was ob- tained f rom Worthington, Nuclease CL 3 f rom Bethesda Research Laboratories, T4-polynucleotide kinase f rom New England Nuclear, T4-RNA ligase f rom Pharmacia . Apyrase (grade III) was purchased f rom Sigma. [y-32P]ATP and [14C]glutamate were

obtained commercially. All other chemicals were o f reagent grade.

Aminoacylation assays

The glutamate acceptor activity o f t R N A samples was determined by charging a limiting amoun t o f t R N A with [14C]glutamate and a barley embryo S-

100 preparation. The s tandard assay for co lumn fractions contained 50 m M Tris-HC1 (pH 7.5), 1 5 m M MgC12, 4 m M DTT, 2 m M ATP, 8 /zM [14C]glutamate (150 mCi /mmole ) , 2070 (v/v) glycer-

ol, 1 m g / m l bovine serum albumin and S-100 prepa-

rat ion (20/zg protein) in a total volume o f 62.5 tzl. Reactions were incubated at 37 °C for 20 min and terminated by spott ing 50/~1 o f each sample onto a W h a t m a n 3MM filter disk (2.3 cm). Filters were im- mediately plunged into ice-cold 5°7o TCA, washed twice more with 5°70 TCA, once with 95°70 ethanol and air dried. Radioactivi ty was determined by scin- tillation counting.

Assay for A L A biosynthesis

Heterologous charging assays for the quant i ta t ion o f plastid t R N A in the samples o f embryo t R N A

were per formed as described [21] with either 4 #g chloroplast enzymes (Blue Sepharose fraction) or 16/~g o f crude embryo S-100 in a total volume o f 15/A, and the amounts o f t R N A specified in Table 1.

Table 1. Determination of the amount of plastid tRNA in a barley embryo tRNA preparation by heterologous charging.

tRNA a Source of synthetase TCA insoluble [14]Glu GIu-tRNA formation Glu accepor activity (cpm/reaction) (pmoles) (pmoles/A260)

- Chloroplast 63 - - Total chloro Chloroplast 12830 31.2 64.2 Glu 2 Chloroplast 311 0.6 1.9 Glul Chloroplast 86 0.05 0.2 Total embryo Chloroplast 1750 4.1 4.1 - Embryo 167 - - Total chloro Embryo 2889 6.6 13.3 Glu 2 Embryo 3165 7.3 2.7 Glu I Embryo 822 1.6 6.1 Total embryo Embryo 6286 14.9 14.9

a Glutamate accepting cytoplasmic tRNA fractions (Glu l, GIu2) were obtained by RPC-5 chromatography (see Fig. 1). Total embryo tRNA was prepared as described in the Methods section. Total chloroplast tRNA was prepared from purified chloroplasts. The amounts of tRNA used in the aminoacylation experiments were: total chloroplast tRNA, 0.5 A260; RPC-5 fractions of cytoplasmic tRNA, 0.25 A260 (Glu 0 or 0.3 A260 (Glu2); total embryo tRNA, 1 A260.

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In order to determine the tRNA specificity of the GIu-tRNA reductase the overall synthesis of ALA was measured starting with precharged [14C]GIu-tRNA as substrate, tRNAs from barley chloroplasts or embryos were glutamylated as described above with the respective homologous synthetase preparation. ALA synthesis was meas- ured essentially as described [8]. The reaction con- tained I mM NADPH, 25 mM MgCI 2, 1 mM DTT, 5 mM levulinate, 0.1 M Tricine-NaOH pH 8.0, ap- proximately 20 #g of protein of Blue Sepharose frac- tion (containing GIuRS and Glu-tRNA reductase), 30 #g of the run-off fraction (containing the aminotransferase) and 5-10 pmoles of [laC]glutamyl-tRNA in a total volume of 0.1 ml. After incubation for 20 min at 28 °C the reactions were terminated by making them 5°70 in SDS, 0.1 M in citric acid and adding 10/xg ALA as a carrier. Af- ter boiling for 2 min, the samples were loaded onto columns of Dowex-50 (Na ÷ -form, 0.75 ml bed vol- ume). Unbound glutamate was washed off with 2 ml of 1 M sodium citrate/25% methanol (pH 3.5) and

3 ml H20. ALA was then eluted with 2 ml of 0.5 M sodium phosphate (pH 6.8). Radioactivity in the fractions was determined after thoroughly mixing the eluates with the 4-fold volume of a water misci- ble liquid scintillator.

Embryo isolation

Embryos were isolated from barley seed according to the procedure of Johnston and Stern [7] including organic solvent flotation. Embryos isolated in this way were largely intact and remained viable for at least one year when stored dry at 4°C.

Isolation and purification of tRNA ctu isoacceptors

Embryos (40 g) were ground in an equal volume of 0.14 M sodium acetate (pH 4.5) and water saturated phenol using a Virtis homogenizer at 45 000 rpm for

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Fig. l . Separation of glutamate accepting tRNA species from barley embryos by reversed-phase chromatography on RPC-5. For details see Materials and Methods.

296

½ min. Crude tRNA was prepared from this extract according to Roe et al. [20]. The crude RNA in the aqueous phase was precipitated by the addition of two volumes of ethanol, tRNA was selectively reco- vered by extracting the resultant precipitate with 1 M NaCl.

The glutamate accepting tRNAs were purified us- ing a combination of RPC-5 chromatography and polyacrylamide gel electrophoresis. The RPC-5 chromatography material was made as described [18] from Plaskon CTFE powder and Adogen 464.

The resulting RPC-5 resin was packed into a column (0.6 x 100 cm) with 10 mM Tris-HC1 (pH 7.0), 10 mM MgC12, 1 mM 2-mercaptoethanol, and 1 M NaCl at 225 psi (approximately 1 ml/min). Stored in this buffer at 4 °C, the resin retains its chromato- graphic resolution for at least one year of regular use. Crude tRNA was fractionated at 20°C with a Pharmacia Liquid Chromatography Controller LCC-500 and two Pharmacia P-500 pumps to pro- vide a linear gradient at the necessary pressure. Crude barley tRNA (182 A254 units) was loaded in the above buffer containing 0.35 M NaCl. After washing the column with 75 ml of the same solution, the tRNA was eluted with a 225 ml gradient of 0.45-0.70 M NaCl followed by a 50 ml NaC1 gra- dient(0.7-1 M) in the above buffer. Fractions of 1.5 ml were collected and subsequently stored at -20 ° C. The glutamate acceptor activity of the RNA in the fractions was determined as described. Active fractions were pooled as indicated in Fig. 1 and each pool was separately fractionated on the same column using a different buffer system. The second RPC-5 fractionation was carried out in the absence of MgCl 2 in 10 mM sodium acetate (pH 4.5), 1 mM 2-mercaptoethanol using the same NaC1 gradient as described above. Active fractions (specific activity about 280 pmoles/A254 unit) were pooled and the RNA was ethanol precipitated prior to polyacryla- mide gel electrophoresis.

Each tRNA TM isoacceptor pool (Fig. 1) was fur- ther fractionated on a denaturing 1507o polyacryla- mide gel containing 8 M urea. The RNA was visual- ized by staining with toluidine blue and eluted as described [11]. Each fraction contained at least four prominent RNA species. Aminoacylation of the RNA contained in these bands demonstrated only

one glutamate-accepting RNA in each fraction (called tRNA~ lu and tRNA2°U).

Labeling and sequencing

Glutamate-accepting, gel purified tRNA species were either 3 ' labeled with [32p]pCp and RNA li- gase or 5 ' labeled with [-y-32p]ATP and T4 poly- nucleotide kinase and then purified on denaturing 2007o polyacrylamide gels and eluted as described with the addition of 20 #g crude E. coli tRNA as car- rier.

Enzymatic RNA sequencing reactions were car- ried out according to ref. 11. Chemical RNA se- quencing was performed, with modifications [25], according to Peattie [19]. Sequences near the labeled ends were obtained by ethanol precipitating the reac- tions twice before resuspending them in 8 M urea without tracking dyes. The samples were elec- trophoresed on 25070 polyacrylamide/8 M urea se- quencing gels until bromophenol blue loaded into an adjacent lane had migrated 7 cm. Total nucleo-

tide analysis of purified tRNAs was performed as described by Nishimura [14]. Modified nucleotides were rechromatographed on PEI-cellulose thin layer plates along with standards as described by Gupta et al. [5]. The locations of the modified bases were determined using the sequencing procedure of Stan- ley and Vassilenko [24].

Results

Purification and sequence analysis of cytoplasmic tRNA Ctu species from barley

The initial reversed-phase (RPC-5) chromatography of the crude barley tRNA yielded three peaks of glutamate acceptor activity. The major species (the first and third peak, see Fig. 1) were purified to homogeneity and then sequenced as described in the Methods section. The nucleotide sequence of tRNA~ lu and tRNA2 °u is given in Fig. 2. Their primary sequences differ only in one position in the anticodon and the T-loop, respectively. The se- quences show 6607o and 7007o homology to tRNA ~lu

t R N A Glu

(Hordeurn vulgare c y t o p l a s m )

AOH c c A

pU -- A C-G C-G G-C U-A C-G G - C U GmtA

G U GGCCC G A A t tl t t A

D ?'C U G mSCCG GG • I I • C TN.," C G

G D ~ I / A G G A U A c _ G A G A U - A C - G G - C G - C

C C

uty A c / v c

- G

Fig. 2. Sequence of the cytoplasmic glutamate tRNAs from barley. The sequence of tRNA~ lu is shown. The differences to tRNA~ ]u are indicated with arrows.

( an t i codon U U C ) f rom Saccharomyces cerevisiae and Schizosaccharomycespombe [10, 27] and 94070

h o m o l o g y to a t R N A ° u ( an t i codon C U C ) f rom Lu- pinus luteus (J. Barciszewski , pe r sona l c o m m u n i c a - t ion) .

Aminoacylation with cytoplasmic and chloroplast extracts

As has been d e m o n s t r a t e d in m a n y cases (e.g., [3])

t R N A s f rom o r g a n d i e s are f requent ly no t a m i n o a -

cy la ted by the co r r e spond ing enzymes f rom the

cy top la sm o f the same o rgan i sm and vice versa. The

same is t rue for the bar ley cy top lasmic t R N A Glu

species which we sequenced. A m i n o a c y l a t i o n ex-

pe r imen t s (Table 1) wi th ba r ley t R N A s and synthe- tases f rom ch lo rop las t s o r cy top l a sm show tha t

G I u R S f rom pur i f i ed ba r ley ch lo rop las t s charges to- tal embryo t R N A to a subs tan t ia l a m o u n t and the

g lu t ama te - accep t ing F P L C frac t ions (Glul and

297

Table 2. Transfer RNA specificity of the Glu-tRNA reductase from barley chloroplasts a.

RNA b Enzyme Glu + ALA ALA Conversion used for (pmoles) (pmoles) (%) precharging

- - 8.6 c 0.42 Chloro total chloro 54 14 26 Cyto Glu 2 embryo 9 0.3 4 Cyto Glu I embryo 5.2 0.15 3 Embryo total embryo 15 1.8 12

a The overall reaction for ALA synthesis was performed with [14]Glu-tRNAs as substrate which were precharged with their respective homologous synthetases (see Materials and methods). The endogenous background was determined by substituting [14]Glutamate for [14]GIu-tRNA; the amount of radioactivity co-eluting with ALA was subtracted from all the tRNA containing reactions. The percentage of conversion of Glu-tRNA to ALA was calculated as the ratio of the amount of radioactivity co-eluting with ALA and the sum of the radio- activity co-eluting with ALA and with glutamate.

b tRNA and enzyme preparations are as specified in Table 1. c This corresponds to a total of 3532 cpm co-eluting with gluta-

mate and ALA.

Glu 2 in Table 1) to a smal l bu t s igni f icant a m o u n t

when c o m p a r e d to the charging o f cy top lasmic

t R N A by cy top lasmic synthetases . In add i t ion ,

t R N A f rom pur i f i ed ch lo rop las t s is aminoacy l a t ed

by c rude embryo synthetases . This suggests tha t the

rest ing embryo con ta ins p rop las t ids capab le o f per-

fo rming pro te in b iosynthes is .

Specificity o f GIu-tRNA reductase for tRNA

The assays for A L A biosynthes i s were p e r f o r m e d

with ba r ley ch lo rop las t enzyme f rac t ions sup-

p l emen ted with t R N A s precharged with [14C]gluta-

ma te by thei r respect ive h o m o l o g o u s synthetases , i.e.

ch lo rop las t o r embryo enzymes. I t is a s sumed tha t

di f ferences in the a m o u n t o f A L A synthesis reflect

the t R N A speci f ic i ty o f the G I u - t R N A reductase. As

Table 2 shows for the ch lo rop las t t R N A s , a b o u t

26°7o o f the i npu t g lu t ama te was conver ted to A L A .

Total ba r l ey embryo t R N A precharged with embryo synthetases gave a convers ion o f 12070. Cons ide r ing

the fact tha t on ly a b o u t ha l f o f the ch lo rop las t

specif ic g lu t ama te accep to r ac t iv i ty is due to

298

tRNA Glu [21, 23] (the remainder is glutamylated tRNA GIn) these numbers agree with the results ob- tained by heterologous charging experiments (Ta- ble 1). We conclude that the GIu-tRNA de- hydrogenase does not recognize the cytoplasmic tRNA alu species, but the small amount of plastid specific tRNA that is present in the total embryo tRNA preparation used for this experiment. Like- wise, the small amount of glutamate converted to ALA in the case of the FPLC purified cytoplasmic tRNA Glu species can be attributed to a slight con- tamination of this preparation with organelle specif- ic tRNA.

Discussion

Synthetase specificity

The sequences of barley cytoplasmic tRNA Glu spe- cies show very little homology to tRNA Glu from chloroplasts [22]. Only parts of the T-stems are simi- lar; overall the homology is less than 54%. Unlike chloroplast tRNA Glu, barley cytoplasmic tRNA Glu

species retain the universally conserved G53-C61 base pair. The lack of homology is not surprising as it is well known that in many cases organellar tRNA species cannot be aminoacylated by the correspond- ing cytoplasmic enzymes and vice versa [3]. There are not enough cytoplasmic tRNA °u species se- quenced to date to allow detailed predictions of the possible determinants for the charging specificity.

Glu-tRNA reductase characteristics

As far as the mechanism of the GIu-tRNA reductase is concerned, it is clear that the tRNA is required for more than a simple activation of the glutamyl moie- ty. Presumably, the enzyme has a specific binding site for tRNA where it is able to discriminate be- tween a large number of structurally different tRNA species. After binding the charged tRNA, the acti- vated glutamate residue could be transacylated onto the enzyme for subsequent reduction while the tRNA is released in order to start a new cycle of glutamylation by the ligase.

Given the unusual tRNA involvement in a reduc- tion reaction we wanted to explore the structural parameters in tRNA which bring about its specifici- ty for serving as a substrate for GIu-tRNA reductase. As the results in this work have shown, the enzyme from barley chloroplasts prefers the chloroplast tRNA Glu over the corresponding isoacceptors found in the cytoplasm. As the tRNA was precharged for this reaction, the specificity of the aminoacyl-tRNA synthetase does not play a role in this reaction. In addition to this study, earlier work has shown that a number of tRNA species do not participate in this reaction in barley chloroplasts [8]. For instance, tRNAs from bacteria, yeasts, and higher plant cytoplasm cannot reconstitute ALA synthesis in the barley system. Upon comparison of the sequences of these tRNAs to the barley chlo- roplast tRNA 61u species [21, 22] one may speculate that the unusual A53- U61 pair may be crucial for in- teraction with the GIu-tRNA reductase. Clearly, in vitro mutagenesis of the appropriate tRNA gene and synthesis of its product will lead to tRNAs which can be used to test this prediction. The tRNA specificity may vary in GIu-tRNA reductases from different or- ganisms; E. coli tRNA 61u is a substrate, albeit a poor one, for the enzyme from Synechocystis 6803 [17] and Chlamydomonas [6]. Both the functional and structural basis of tRNA Glu recognition by the GIu-tRNA reductase remain unknown. Due to the lack of GlnRS in barley chloroplasts [21] there are two chloroplast glutamyl-tRNA pools, Glu- tRNA °u and Glu-tRNA GIn. While the pool of Glu- tRNA GIn in barley chloroplasts is limited, the strict tRNA specificity of the Glu-tRNA reductase may reflect a need to protect this pool from depletion by ALA synthesis under conditions of rapid chlo- rophyll biosynthesis. The relevance of the unusual A53.U61 base pair found in all chloroplast glutamate-acceptor tRNAs sequenced to date, as well as in the tRNA Glu from Synechocystis [17] can be explored by introducing a similar sequence into E. coli tRNA2 Glu in place of its universal G53.C61. Likewise, barley mitochondrial tRNA Glu, as yet un- sequenced, may provide more clues to the identity of the sequences or structures required for the recogni- tion of tRNA by the Glu-tRNA reductase.

Acknowledgements

Barley chloroplast enzymes were a gift of C. G. Kan- nangara, Carlsberg Laboratory, Copenhagen. This work was supported by grants from the Department of Energy and from the National Science Founda- tion.

References

1. Beale SI: A-aminolevulinic acid in plants: its biosynthesis, regulation, and role in plastid development. Ann Rev Plant Physiol 29:95-120 (1978).

2. Beale SI, Gough SP, Granick S: Biosynthesis of 6- aminolevulinic acid from the intact carbon skeleton of glu- tamic acid in greening barley. Proc Natl Acad Sci USA 72: 2719-2723 (1975).

3. Burkard G, Guillemaut P, Weil JH: Comparative studies on the tRNAs and the aminoacyl-tRNA synthetases from the cytoplasm and the chloroplast of Phaseolus vulgaris. Bi- ochim Biophys Acta 224:184-198 (1970).

4. Friedmann HC, Thauer RK: Ribonuclease-sensitive 6- aminolevulinic acid formation from glutamate in cell extracts of Methanobacterium thermoautotrophicum. FEBS Lett 207:84-88 (1986).

5. Gupta RC, Randerath E, Randerath K: An improved separa- tion procedure for nucleoside monophosphates on PEI plates. Nucleic Acids Res 3:2915-2921 (1976).

6. Huang DD, Wang W-Y: chlorophyll biosynthesis in Chlamydomonas starts with the formation of glutamyl- tRNA. J Biol Chem 261:13451-13455 (1986).

7. Johnston FB, Stern H: Mass isolation of viable wheat em- bryos. Nature 179:160-161 (1957).

8. Kannangara CG, Gough SP, Oliver RP, Rasmussen SK: Bi- osynthesis of 6-aminolevulinate in greening barley leaves V1. Activation of glutamate by ligation to RNA. Carlsberg Res Comm 49:417-437 (1984).

9. Kannangara CG, Schouboe A: Biosynthesis of 6- aminolevulinate in greening barley leaves. VII. Glutamate 1- semialdehyde accumulation in gabaculine treated leaves. Carlsberg Res Comm 50:179-191 (1985).

10. Kobayashi T, Irie T, Yoshida M, Takeishi K, Ukita T: The primary structure of yeast glutamic acid transfer RNA specif- ic to the GAA codon. Biochim Biophys Acta 366:168-181 (1974).

11. Krupp G, Gross H J: Sequence analysis of in vitro [32p]_ labeled RNA. In: Agris PF, Kopper RA (eds) The Modified Nucleosides in Transfer RNA II: A Laboratory Manual of Genetic Analysis, Identification and Sequence Determina- tion. Alan R. Liss Inc., New York (1983) pp. 11-58.

299

12. Mau Y-H, Wang W-Y, Tamura RN, Chang T-E: Identifica- tion of an intermediate of 6-aminolevulinic acid biosynthesis in Chlamydomonas by HPLC. Arch Biochem Biophys 255: 75-79 (1987).

13. McKie J, Lucas C, Smith A: A-aminolaevulinate biosynthesis in the cyanobacterium Synechococcus 6301. Phytochem 20: 1547-1549 (1981).

14. Nishimura S: Chromatographic mobilities of modified nucleotides. In: Schimmel P, S611 D, Abelson JN (eds) Trans- fer RNA: Structure, Properties and Recognition. Cold Spring Harbour Laboratory, New York (1979) pp. 551-552.

15. Oh-Hama T, Seto H, Miyachi S: 13C NMR evidence for bac- teriochlorophyll c formation by the C5 pathway in the green sulfur bacterium Prosthecochloris. Eur J Biochem 159: 189-194 (1986).

16. Oh-Hama T, Seto H, Miyachi S: 13C-NMR evidence of bac- teriochlorophyll a formation by the C5 pathway in Chromati- um. Arch Biochem Biophys 246:192-198 (1986).

17. O'Neill GP, Peterson DM, Sch6n A, Chen M-W, S611 D: For- mation of the chlorophyll precursor 6-aminolevulinic acid in cyanobacteria requires aminoacylation of a tRNAGIu spe- cies. Manuscript submitted.

18. Pearson RL, Weiss JF, Kelmers AD: Improved separation of transfer RNA's on polychlorotrifluoroethylene supported reversed phase chromatography columns. Biochim Biophys Acta 228:770-774 (1971).

19. Peattie D: Direct chemical method for sequencing RNA. Proc Natl Acad Sci USA 76:1760-1764 (1979).

20. Roe B, Marcu K, Dudock B: The isolation and sequence anal- ysis of transfer RNA: The use of Plaskon chromatography (RPC-5). Biochim Biophys Acta 319:25-36 (1973).

21. Sch6n A, Kannangara CG, Gough S, S611 D: Protein bi- osynthesis in organelles requires misaminoacylation of tRNA. Nature 331:187-190 (1988).

22. Sch6n A, Krupp G, Gough S, Berry-Lowe S, Kannangara CG, S611 D: The RNA required in the first step of chlorophyll bi- osynthesis is a chloroplast glutamate tRNA. Nature 322: 281-284 (1986).

23. Sch6n A, S611 D: tRNA specificity of a mischarging aminoacyl-tRNA synthetase: glutamyl-tRNA synthetase from barley chloroplasts. FEBS Lett 228:241-244 (1988).

24. Stanley J, Vassilenko S: A different approach to RNA se- quencing. Nature 274:87-89 (1978).

25. Waldmann R, Gross H J, Krupp G: Protocol for rapid chemi- cal RNA sequencing. Nucleic Acids Res 15:7209 (1987). (1987).

26. Wang W-Y, Huang D-D, Stachon D, Gough SP, Kannangara CG: Purification, characterization, and fractionation of the &aminolevulinic acid synthesizing enzymes from light- grown Chlamydomonas reinhardtii cells. Plant Physiol 74: 569-575 (1984).

27. Wong T-W, McCutchan T, Kohli J, $611 D: The nucleotide sequence of the major glutamate transfer RNA from Schizosaccharomyces pombe. Nucleic Acids Res 6: 2057-2068 (1979).