expression escherichia trna genesfrom promoter the … · ers (23). theexistence ofaminorinternal...

Vol. 166, No. 1JOURNAL OF BACTERIOLOGY, Apr. 1986, p. 306-3120021-9193/86/040306-07$02.00/0Copyright C 1986, American Society for Microbiology

Expression in Escherichia coli of Bacillus subtilis tRNA Genes froma Promoter within the tRNA Gene Region

BARBARA S. VOLD* AND CHRISTOPHER J. GREENDepartment of Molecular Biology, SRI International, Menlo Park, California 94025

Received 15 April 1985/Accepted 30 December 1985

A cloned DNA segment from Bacillus subtilis containing 21 tRNA genes was introduced into Escherichia cQli.In the B. subtilis genome, these tRNA genes are locatted after an rRNA gene set and before tandem terminators.The rRNA and tRNA genes are thought to represent a single transcriptiqnal unit. However, another putativepromoter occurs after the second tRNA gene within the tRNA gene cluster and has a sequence compatible withboth the major B. subtilis (sigma 43 type) promoter and the major E. coli promoter. The B. subtilis21-tRNA-gene cluster was introduced into E. coli to see whether this promoter would be recognized in E. coli,to determine the start point of transcription in the E. coli system, and to see whether mature B. subtilis tRNAswould be transcribed and processed in E. coli. Expression was evaluated by monitoring levels of aminoacylationof mature tRNAs extracted from E. coli containing plasmids with or without the B. subtilis tRNA genes and byexamining profiles of isoaccepting species on columns of RPC-5. Si nuclease mapping was performed to definethe starting point for transcription. The results indicated that a putative promoter located within the R. subtillstRNA gene region was functional when cloned into E. coli and that it initiated at the same nucleotide as it doesin B. subtilis. In addition, at least some B. subiilis tRNA genes could be transcribed and processed in E. colito mature tRNAs capable of accepting an amino acid.

Information about sequence analysis and organization oftRNA genes of Bacillus subtilis has been advancing sorapidly in the past few years that there are now more knowntRNA gene sequences from B. subtilis than from Escherichiacoli. Despite all this information, few transcriptional analy-ses have been done. The tRNA gene organization in B.subtilis differs from that in E. coli in ways that may affecttRNA gene transcription and processing. For instance,tRNA genes in B. subtilis tend to be highly clustered, withthe largest group containing 21 genes, whereas the largestknown unit in E. coli contains 7 tRNA genes (5, 16). Mostknown tRNA gene clusters in B. subtilis follow rRNA genesets. Although the sequences are probably transcribed aspart of the entire rRNA-tRNA unit, inspection of B. subtilisDNA regions containing tRNA genes indicates that theremay be promoter elements distal to the dual rRNA promot-ers (23). The existence of a minor internal promoter betweentRNA genes in one polycistronic operon of E. coli has beenpostulated (12). However, none of the other translationalunits in which tRNA genes are found in E. coli havepromoters within the tRNA genes (5).Here we describe the introduction of a cluster of 21 B.

subtilis tRNA genes into E. coli both to investigate theability of a putative promoter element to function within thetRNA gene region and to determine whether these genes areexpressed in the E. coli system.

MATERIALS AND METHODSPlasmids. A 3.3-kilobase PstI-EcoRI DNA segment from B.

subtilis containing the 3' end of 23S rRNA, a 5S rRNA, and21 tRNA genes was cloned into pUC8. The sequence of thisDNA segment has been previously described (10). Theconstruct, designated pUCTG5, was cloned in a directionopposite to that ofthe P-galactosidase promoter ofpUC8. Forcloning into pMK3 (kindly provided by Ron Yasbin [21]), a

* Corresponding author.

3.0-kilobase insert in pUCTG5 was excised with XmaI andsubcloned into pMK3; it was designated pMKTG.Growth of cells and extraction of tRNA. E. coli JM103 and

JM101 were used as hosts for pUC8 and pMK3, respec-tively. Cells were transformed by the procedure of Cohen etal. (4) and were grown to exponential growth phase in 2x YTmedium (16 g of tryptone, 10 g of yeast extract, and 5 g ofNaCl per liter). tRNA was removed from freshly preparedcells by direct phenol extraction (24) and was further purifiedby. elution from DEAE-cellulose.

Aminoacylation reactions and resolution of isoacceptor spe-cies by column chromatography on RPC-5. tRNAs wereaminoacylated and fractionated on RPC-5 by using previ-ously described procedures (22, 24). All aminoacylationreactions were done with a mixed aminoacyl-tRNA synthe-tase preparation from B. subtilis under conditions that weremaximized for aminoacylation of B. subtilis tRNAs (24).Thus, although the conditions may not have been optimal forE. coli tRNAs, they favored the detection of B. subtilistRNAs.

Purification and sequence analysis of tRNAs. tRNAs wereextracted from E. coli JM103 carrying pUCTG5, fraction-ated on a large column of RPC-5, identified by postchargingprocedures, rechromatographed on a small column ofRPC-5, and fractionated further by column chromatographyon Aminex A-28 (Bio-Rad Laboratories, Richmond, Calif.).These partially purified tRNA fractions were then labeled atthe 3' terminus with [5'-32P]cytidine 3',5'-bis(phosphate),fractionated by electrophoresis on a 15% polyacrylamide-7M urea gel, eluted, and partially sequenced by the chemicalsequencing technique described by Peattie (17).

S1 nuclease mapping. RNAs for Si nuclease mappingexperiments were extracted by the procedure of Aiba et al.(1). The single-stranded DNA probe was made in vitro froma 314-base-pair HpaII fragment of the B. subtilis 21-tRNAgene cluster cloned into the AccI site of bacteriophageM13mp7. This cloned fragment begins at the end of thetRNAThr gene and ends at the beginning of the tRNA UAA

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B. SUBTILIS tRNA GENES EXPRESSED IN E. COLI 307

TABLh 1. Amirloacylation levels of tRNAs isolated froniE. coli cells containing a plasmid with or without an insert

containing 21 B. subtilis tRNA genes

Relative aminoacylation levelaAmino acid

Controlb pUCTG5 Control pMKTG

tRNA genes before internalpromoterVal 1.11 0.61 2.07 1.66Thr 0.45 0.47 0.81 0.62

tRNA genes followinginternal promoterLys 1.11 6.76 2.05 5.51Leu 0.67 6.39 1.37 2.38Gly 1.79 5.32 3.24 8.44LeucArg 0.67 6.20 1.41 4.75Pro 0.93 4.18 1.01 2.25Ala 1.59 2.17 2.17 2.00Met 1.47 2.35 2.09 3.44Ile 0.76 1.59 2.31 2.48Ser 1.07 0.69 1.41 1.40MetcAsp 1.11 1.25 1.76 1.82Phe 1.13 2.00His 0.38 0.56 0.49 0.30GlycIlecAsn 1.75 2.09 2.10 1.68SercGlu

tRNA genes not on insertTyr 1.00 1.00 1.00 1.00Trp 0.40 0.70 0.25 0.33a Values for a particular amino acid-accepting group relative to tyrosine.

There is no tyrosine gene on the DNA insert, so the tyrosine activity shouldrepresent that from E. coli tRNAs.

b Control tRNAs from E. coli grown with pUC8 in strain JM103 or pMK3in JM1O1.

c Although no tRNA gene sequences are repeated within this cluster, thereare cases in which two tRNA genes within the cluster belong to the sameamino acid-acceptor group. For cases such as this (i.e., Leu), the data aregiven the first time one isoacceptor appears.

gene (10) (Fig. 1). The orientation of this clone resulted in theproduction of a single-stranded DNA that was the samesense as the putative tRNA precursor. A DNA primner 20bases long, complementary to the first 20 bases of the first

tRNALeu gene, was synthesized on an Applied Biosystemsmodel 380A DNA synthesizer. This primer was used toproduce a sequencing ladder by the dideoxy chain termina-tion technique with [a-32P]dCTP (600 to 800 Ci/mmol; New England Nuclear Corp., Boston, Mass.) (13, 19).The primer was also used to produce a 32P-labeled probecomplementary to the tRNA precursor by using the samesequencing reactions in the absence of dideoxynucleotides.This resulted in the synthesis of a long labeled DNA strandbase paired to the M13mp7 template. This double-strandedproduct was cleaved at the EcoRI site downstream from theAccI-HpaII junction on M13mp7. The labeled probe waspurified on an 8% acrylamide-7 M urea gel to remove boththe M13mp7 template and the large labeled fragments thatwere synthesized from the M13mp7 region. The labeledprobe was cut out of the gel, crushed in TE buffer (10 mMTris, 1 mM EDTA [pH 7.5]), and eluted overnight at 5°C.This DNA was then purified on an NENsorb column (NewEngland Nuclear) and used as the single-stranded DNAprobe for Si nuclease mapping by the procedure of Gilmanand Chamberlin (7). The products of the Si digestion wereelectrophoresed on a 5% acrylamide-7 M urea gel alohgsidea dideoxy sequencing of the M13mp7 template (13, 19) byusing the same 20mer primer that was used to generate thelabeled probe.

RESULTSAminoacylation levels. Levels of aminoacylation were

measured for several amino acceptor groups of tRNAsisolated from E. coli cells grown to exponential phase whichcarried a plasmid with or without an insert containing 21 B.subtilis tRNA genes (Table 1). The insert (Fig. 1) containeda potential promoter region characteristic of promoters fromE. coli (18, 20) and B. subtilis (14, 23). The -35 sequencewas inside the tRNAThr gene. In addition, this sequencecontained a region suggested by Ogasawara et al. (15) to beassociated with genes in B. subtilis that are under stringentcontrol. The amino acid acceptor groups are listed in theorder, from top to bottom, in which they occurred on theinsert (Fig. 1). The genes for valine and threonine tRNAsoccurred before the internal promoter. The two amino acidacceptor groups for tyrosine and tryptophan did not havetRNA genes represented in this B. subtilis tRNA gene insert.Therefore, those genes must have been expressed from theE. coli genome and served as controls. Because the tRNApreparations differed in specific activity, the data werenormalized to levels of tyrosyl-tRNA (Table 1). High levels

33 310 5 14 9 15 5 18 2 6 17 11 12 16 10 15 10 3 25

Val Thr Lys Leu Gly Leu Arg Pro Ala Met lle Ser fMet Asp Phe His Gly lIe Asn Ser GluS ~~~~~~~~~~~~~(Met)

tRNAThr t NA YS

.... CTCTTGCCGGCACCACTTTTATATGATATAATATTCAA.GTCTATTGTAAGAAGAGCCATTAG....-35 -10 Stringent

FIG. 1. Schematic representation of cloned DNA segment showing the location and sequence of putative transcriptional control signalsand the order of tRNA genes. HpaII sites (CCGG) at the end of the tRNAThr gene and at the beginning of the first tRNALeU gene were usedto subclone the region containing the putative transcription start site.

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308 VOLD AND GREEN

ISOACCEPTOR CLASSES NOT ON INSERT:

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FIG. 2. RPC-5 column chromatography of tRNAs extracted from E. coli cells carrying pUC8 with or without the tRNA gene insert.Profiles are shown for two aminoacyl-tRNA classes not found in this tRNA gene cluster (tyrosine and tryptophan) and then foraminoacyl-tRNA classes found on the plasmid, arranged in the order in which they first occur in the tRNA gene cluster. Counts per minutefor both 3H- and '4C-amino acids are given on the y axis; therefore, the counts per minute for 14C are usually multiplied by some constant as

noted. The isoacceptor group is shown at the bottom of the graph. tRNAs from cells containing pUCTG5 were aminoacylated with a 3H-aminoacid, and tRNAs from cells with pUC8 were aminoacylated with a "4C-amino acid. x, Tritium; A, 14C.

of expression were detectable only from the first six genesoccurring after the internal promoter. The aminoacylationlevels from E. coli overproducing foreign tRNAs may havebeen affected by a lack of complete modification (6) or by anoverloading of the processing system; however, a generaltrend in aminoacylation values seemed evident.Chromatography of tRNAs from E. coli grown with pUC8

or pUCTG5. Resolution of isoaccepting species on RPC-5was performed by aminoacylating the tRNA preparation(from cells grown with pUCTG5) with a 3H-amino acid andby aminoacylating the control tRNA preparation (from cellsgrown with pUC8) with a '4C-amino acid. After mixing, the

aminoacyl-tRNAs were fractionated on RPC-5 (22). RPC-5profiles were examined (Fig. 2). As mentioned in the Mate-rials and Methods section, pUCTG5 had the B. subtilistRNA gene region inserted in an orientation opposite to thatof the ,-galactosidase promoter of pUC8, so that any tran-scription must have been initiated within the inserted region.Profiles were grouped in three sections: tRNAs from isoac-ceptor classes not occurring on the insert, tRNAs fromisoacceptor classes occurring within the insert but before theinternal putative promoter, and tRNAs from isoacceptorclasses occurring after the internal promoter.The two tRNAs from isoacceptor classes not occurring in

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310 VOLD AND GREEN

the inert and the two tRNAs from classes before the internalpromoter showed no major differences between isoacceptorsfrom E. coli cells grown with the plasmids with or withoutthe B. subtilis tRNA genes. In the profiles for the 13isoacceptor classes occurring after the internal promoter, wecould easily resolve detectable differences in all but fourcases. Therefore, we consider it significant that no differ-ences in profiles were detected in the two control classes,although it is possible that new species occurred that fortu-itously cochromatographed with existing species.

Sequence analysis of mature tRNAs. Differences in profilescould have been caused by the presence of new isoacceptorswith different sequences or by differences in posttranscrip-tional modification of tRNAs having the same primarystructure. To eliminate the possibility that these new specieswere improperly modified E. coli tRNAs, sequence analysiswas done on the tRNAs from three isoaccepting groups:glycine, arginine, and phenylalanine. Sequence analysisshowed that the late-eluting peak for glycine tRNA frompUCTG5 (Fig. 2), the slightly later-eluting peak for phenyl-alanine from pUCTG5, and the late-eluting arginyl-tRNAshad primary sequences of B. subtilis tRNAs. We did notinvestigate the state of modification of these B. subtilistRNAs.With respect to the order of tRNA genes on this particular

tRNA gene set, glycine tRNA genes were at positions 3 and15. The sequence of the late-eluting B. subtilis glycyl-tRNAwas that found in position 3. The arginine tRNA gene was atposition 5, and phenylalanine was at position 13.

Detailed mapping of the transcription initiation site. Theorientation and approximate location of the putative pro-moter region is shown in Fig. 1. This region was indicated tobe a promoter region in the in vivo expression studiesdescribed above. To demonstrate that this was the promoterregion, the start point of transcription was determined by S1nuclease mapping. The hybridization and Si nuclease pro-tocols were those used by Gilman and Chamberlin (7) basedon the procedure of Berk and Sharp (2). The single-strandedDNA probe for the B. subtilis tRNA gene region coveringthe putative promoter region was made by using a syntheticprimer from B. subtilis DNA cloned into phage M13mp7.This single-stranded probe was hybridized to unlabeledRNA isolated from E. coli cells which harbored the plasmidpUC8 or the plasmid with B. subtilis tRNA genes, pUCTG5.In addition, the probe was hybridized to RNA extractedfrom exponentially growing cells of B. subtilis. After hybrid-ization, nonhybridized, single-stranded nucleic acid wasdigested with S1 nuclease, and the resulting protected hy-brids were denatured and analyzed by electrophoresisthrough polyacrylamide alongside a dideoxy sequencingladder of the same region initiated with the same syntheticprimer (Fig. 3). Thus, the protected fragments comigrated toa point which could be precisely located in the sequence.A protected fragment (indicated by the arrow labeled with

an S [Fig. 3]) locates the start point of transcription. Theexpanded region (Fig. 3) gives the sequence of that regionand indicates which nucleotide in the sequence is the startsite. The location of this start site has also been indicated inFig. 1. All three preparations of RNA from E. coli harboringpUCTG5 produced this protected fragment (Fig. 3, lanes 7,9, and 10). No fragment of this size was protected in thecontrol. The next largest protected fragment (Fig. 3, lanes 7,9, and 10) was 15 bases smaller and may have represented anRNase P-cleaved product composed of the 5' end of thetRNALYS transcript with an unprocessed 3' end. We inter-preted the series of smaller fragments as being due to

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1 2 3 4 5 6 7 8 9 10FIG. 3. Analysis of fragments protected from Si nuclease. A 5%

acrylamide-7 M urea gel was used to determine the location offragments protected from Si relative to the sequence of the pro-moter region. The photograph is a composite from two exposures ofthe same autoradiogram to equalize the darkness of the dideoxysequencing fragments and that of the fragments protected from Si.Lane 1, 12P-labeled antisense DNA probe before Si nucleasedigestion. Lanes 2 through 5, G, A, T, and C dideoxy sequencinglanes produced from the same primer and template as those used togenerate the probe. Lane 6, The probe was incubated with RNAfrom E. ccli JM101 with a pUC8 plasmid with no insert. Lanes 7, 9,and 10, Protected fragments generated by incubating the DNA probewith RNA from E. coli containing pUCTG5; in the experimentrepresented in lane 10, the cells were incubated with 170 p.g ofchloramphenicol per ml 20 min before harvesting. Lane 8, RNAextracted from exponentially growing B. subtilis 168. 5, Transcrip-tion start site; B, Hpall-Accl junction and the beginning of the B.subtilis sequence; L, 5' processing site for lysine tRNA. Theexpanded sequence on the left represents the sequence complemen-tary to the RNA transcript.

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intermediates in the 3' end processing of the tRNALYS. Anyfragment protected by mature B. subtilis tRNALYS wouldhave been found near the bottom of the gel (Fig. 3, lane 8).RNA isolated from B. subtilis was also examined (Fig. 3).

A fragment was detected that electrophoresed to the samelocation as the protected fragment in the E. coli host thatharbored the B. subtilis 21-tRNA-gene insert. This indicatedthat this start site was also used in normal B. subtilis cells.The highest-molecular-weight fragment protected by RNAfrom normal B. subtilis cells (Fig. 3, lane 8) was interpretedas containing all of the sequence in the synthetic probe fromB. subtilis. This represented transcription from a promoterupstream from the one that occurred within the tRNAcluster. The B. subtilis tRNA gene cluster followed therRNA operon rrnB, the DNA sequence of which has beencompleted (9). No identifiable terminator sequences oc-curred between the dual rRNA promoters and the tRNAgenes. Thus, the presence of a protected fragment higher inmolecular weight than that protected by the transcript fromthe internal tRNA gene promoter may have representedread-through from the rRNA promoters. This highest-molecular-weight fragment was not found in the E. coli cellscontaining pUCTG5 because the rRNA promoters were notpresent in that insert (Fig. 1). The mapping of this region atvarious stages of development in B. subtilis is under inves-tigation.

DISCUSSION

These studies showed that a putative promoter that oc-curred within a cluster of 21 B. subtilis tRNA genes wasfunctional in a B. subtilis fragment that was introduced intoE. coli on a multicopy plasmid. The sequence of thispromoter was compatible with the consensus sequence for asigma 43 type promoter from B. subtilis (14) and the major E.coli promoter (18, 20). Elements outside the -10 and -35consensus sequences have been shown to be important forthe transcription of tRNA species (11); therefore, the corre-spondence in consensus sequences was not necessarilysufficient to ensure that tRNA genes from a large clusterfrom B. subtilis would be transcribed and processed.

Previously reported increases in aminoacylation for mo-nomeric or dimeric E. coli tRNA genes cloned into E. coliare around two- to ninefold (3, 8, 20). The tRNA genes closeto the promoter on the B. subtilis tRNA gene segment fellinto this range; however, the levels of aminoacylation de-creased for tRNA species from the more distal tRNA genes.In E. coli, the transcription of stable RNAs does not exhibitpolarity (8); consequently, this apparent polarity effect mightbe due to the fact this was a heterologous system in whichsome aspect of transcription, processing, or both was aber-rant. Nevertheless, mature tRNAs having the primary se-quence of B. subtilis tRNAs can be transcribed and proc-essed to species capable of accepting an amino acid, whichemphasized the overall similarities between the two eubacte-rial systems.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grant GM29231 from the National Institutes of Health.Annette Wen, Barry Wong, Lewis Kraus, Ping Sze, and Kathleen

Okamoto are acknowleged for technical assistance.

LITERATURE CITED1. Aiba, H., S. Adhya, and B. de Crombrugghe. 1981. Evidence for

two functional gal promoters in intact Escherichia coli cells. J.Biol. Chem. 256:11905-11910.

2. Berk, A. J., and P. A. Sharp. 1977. Sizing and mapping of earlyadenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12:721-732.

3. Caillet, J., J. Plumbridge, M. Springer, J. Vacher, C.Delamarche, R. Buckingham, and M. Grunberg-Manago. 1983.Identification of clones carrying an E. coli tRNAPhC gene bysuppression of phenylalanyl-tRNA synthetase thermosensitivemutants. Nucleic Acids Res. 11:727-736.

4. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Non-chromosomal antibiotic resistance in bacteria: genetic transfor-mation of Escherichia coli by R-factor DNA. Proc. Natl. Acad.Sci. USA 69:2110-2114.

5. Fournier, M. J., and H. Ozeki. 1985. Structure and organizationof the transfer ribonucleic acid genes of Escherichia coli K-12.Microbiol. Rev. 49:379-397.

6. Gefter, M. L., and R. L. Russell. 1969. Role of modifications intyrosine transfer RNA-a modified base affecting ribosomebinding. J. Mol. Biol. 39:145-157.

7. Gilman, M. Z., and M. J. Chamberlin. 1983. Developmental andgenetic regulation of Bacillus subtilis genes transcribed bysigma28-RNA polymerase. Cell 35:285-293.

8. Gourse, R. L., and M. Nomura. 1984. Level of rRNA, nottRNA, synthesis controls transcription of rRNA and tRNAoperons in Escherichia coli. J. Bacteriol. 160:1022-1026.

9. Green, C. J., G. C. Stewart, M. A. Hollis, B. S. Vold, and K. F.Bott. 1985. Nucleotide sequence of the Bacillus subtilis ribo-somal RNA operon, rrnB. Gene 37:261-266.

10. Green, C. J., and B. S. Vold. 1983. Sequence analysis of acluster of twenty-one tRNA genes in Bacillus subtilis. NucleicAcids Res. 11:5763-5774.

11. Lamond, A. I., and A. A. Travers. 1983. Requirement for anupstream element for optimal transcription of a bacterial tRNAgene. Nature (London) 305:248-250.

12. Lee, J. S., G. An, J. D. Friesen, and N. P. Fiil. 1981. Location ofthe tufB promoter of E. coli: contranscription of tufB with fourtransfer RNA genes. Cell 25:251-258.

13. Messing, J., R. Crea, and P. H. Seeburg. 1981. A system forshotgun DNA sequencing. Nucleic Acids Res. 9:309-321.

14. Moran, C. P., Jr., N. Lang, S. F. J. LeGrice, G. Lee, M.Stephens, A. L. Sonenshein, J. Pero, and R. Losick. 1982.Nucleotide sequences that signal the initiation of transcriptionand translation in Bacillus subtilis. Mol. Gen. Genet. 186:339-346.

15. Ogasawara, N., S. Moriya, and H. Yoshikawa. 1983. Structureand organization of rRNA operons in the region of the replica-tion origin of the Bacillus subtilis chromosome. Nucleic AcidsRes. 11:6301-6318.

16. Ozeki, H. 1980. The organization of transfer RNA genes inEscherichia coli, p. 173-183. In S. Osawa, H. Ozeki, H. Uchida,and T. Yura (ed.), Genetics and evolution of RNA polymerase,tRNA and ribosomes. University of Tokyo Press, Tokyo.

17. Peattie, D. A. 1979. Direct chemical method for sequencingRNA. Proc. Natl. Acad. Sci. USA 76:1760-1764.

18. Rosenberg, M., and D. Court. 1979. Regulatory sequencesinvolved in the promotion and termination of RNA transcrip-tion. Annu. Rev. Genet. 13:319-353.

19. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

20. Schwartz, I., R.-A., Klotsky, D. Elseviers, P. J. Gallagher, M.Krauskopf, M. A. Q. Siddiqui, J. F. H. Wong, and B. A. Roe.1983. Molecular cloning and sequencing of pheU, a gene forEscherichia coli tRNAPhe. Nucleic Acids Res. 11:4379-4389.

21. Sullivan, M. A., R. E. Yasbin, and F. E. Young. 1984. Newshuttle vectors for Bacillus subtilis and Escherichia coli whichallow rapid detection of inserted fragments. Gene 29:21-26.

22. Vold, B. S. 1973. Analysis of isoaccepting transfer ribonucleicacid species of Bacillus subtilis: chromatographic differences

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between transfer ribonucleic acids from spores and cells inexponential growth. J. Bacteriol. 113:825-833.

23. Vold, B. S. 1985. Structure and organization of genes fortransfer ribonucleic acid in Bacillus subtilis. Microbiol. Rev.49:71-80.

24. Vold, B. S., and S. Minatogawa. 1972. Characterization ofchanges in transfer ribonucleic acids during sporulation inBacillus subtilis, p. 254-263. In H. 0. Halvorson, R. Hanson,and L. L. Campbell (ed.), Spores V. American Society forMicrobiology, Washington, D.C.

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