THE JOURNAL OF BIOLOGwAL CHEmITRYVoL 257, No. 18, Issue of September 25, pp. 11113-11120, 1982Printed in U.S.A.

In Vitro Transcription of the supB-E tRNA Operon of Escherichia coliCHARACTERIZATION OF TRANSCRIPTION PRODUCTS*

(Received for publication, April 12, 1982)

Noboru Nakajima, Haruo Ozeki, and Yoshiro ShimuratFrom the Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan

The seven tRNA genes clustered in the supB-E regionof the Escherichia coli chromosome were transcribed invitro with purified RNA polymerase, using a restrictionfragment from Xpsu 0 2, a transducing phage carryingthe chromosome region, as template. A single majortranscript was synthesized, which was about 770 nu-cleotides long and contained all seven tRNA sequences.The terminal sequences of the transcript were deter-mined and mapped on the DNA sequence of the supB-Eregion previously determined. The transcription startsite is seven base pairs downstream from the Pribnowbox sequence, as expected from the DNA sequenceanalysis and consistent with the findings on the tri-meric tRNA precursor (pppG-tRNAMet-tRNALu -

tRNAG') which was detected in an RNase P mutant andshown to be coded for by the supB-E region. Cleavageof the restriction fragment at the -35 region with an-other restriction endonuclease abolished the templateactivity of the fragment. Transcription of the supB-EtRNA operon was relatively unaffected by the presenceof p factor. Transcription termination occurs within aregion of three bases between positions 770 and 772from the transcription start site. Immediately upstreamfrom the termination sites, there is a region of 26 nu-cleotides that could form a stem structure, therebyconsistent with the general feature of p-independenttermination sites.

Recent advances in nucleotide sequence analysis as well asrecombinant DNA technology have provided much informa-tion on the organization and structure of tRNA genes invarious organisms. However, considerably less is known aboutthe regulation of expression of tRNA genes in any organisms.In Escherichia coli, tRNA genes are distributed throughoutthe genome as part of ribosomal RNA operons or as clusterscontaining either different gene species or repeats of the samegenes (for review, see Ref. 1). As the number of tRNA mole-cules in E. coli does not necessarily correspond to the copynumber of tRNA genes, it is generally assumed that transcrip-tion and post-transcriptional processing reactions may playroles in the regulation of tRNA gene expression. Transcriptionof tRNA genes is regulated, like any other genes, by thetranscription signals such as promoters and terminators. Agreat deal of our knowledge concerning the transcriptionsignals of tRNA genes has come from the studies on the tyrTgene, which codes for tRNAT r (2-8). Transcription termina-

* This work was supported by a Grant-in-Aid for Scientific Re-search from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be herebymarked "advertisement" in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

i To whom correspondence should be sent.

tion of the tyrT gene cluster has been shown to be affected bythe presence of p factor both in vitro and in vivo (3, 8). It isnot known, however, whether p-dependent transcription ter-mination can be generalized to other E. coli tRNA genes.

The supB-E region mapped at 15 min on the E.coli chro-mosome has been shown to contain seven tRNA genes: twotRNA" et genes, two tRNAIn" genes, two tRNA2'n genes, andthe gene coding for an unidentified tRNAX (9-11). This findingwas based on the analyses of a X transducing phage carryingthe supB-E region as well as its suppressor derivatives. Sub-sequently, the complete nucleotide sequences of the supB-Eregon and its flanking regions have been determined (12). Thesequence analysis has revealed that the seven tRNA genes aretightly clustered in the chromosome region and that theorganization of the gene cluster is (5')-tRNAet-9 basepairs-tRNAX-23 base pairs-tRNAGl'-34 base pairs-tRNAl"n-15 base pairs-tRNAMet-47 base pairs-tRNA2in-37base pairs-tRNA2I n. The unidentified tRNA (tRNAx) wasshown to be a new species of tRNA of an anticodon sequence(5'-UAG) corresponding to a leucine codon. Therefore, thistRNA has been tentatively designated tRNALe ". These tRNAsequences are preceded by a heptameric sequence (5'-CATAATG) that is consistent with the Pribnow box sequence(13-17) and by a hexanucleotide TTGACG sequence that islocated further upstream and consistent with the consensussequence of the -35 region, a part of the general RNApolymerase recognition signals (16, 17). On the basis of thesesequences and also of the findings on the trimeric tRNAprecursor containing pppG-tRNAmet-tRNALe"-tRNAGlnwhich was enriched in a temperature-sensitive mutant ofRNase P upon infection of a transducing phage carrying thesupB-E region (18), the putative transcription start site hasbeen surmised (12). It has also been suggested that the seventRNA genes in the supB-E region are controlled by a singlepromoter and thus constitute an operon (12). However, wehave not observed any tRNA precursor that contains theseven tRNA sequences in vivo, although a tetrameric precur-sor containing tRNA -tRNA -tRNAl -tRNA and a tri-meric precursor containing tRNA!e t and tRNA2G' sequenceshave been detected in the RNase P mutant (18). Apparently,the analyses of in vivo transcription products were hamperedby the instability of nascent RNA molecules due to rapidprocessing reactions. On the other hand, very little is knownabout the site(s) of transcription termination of the tRNAoperon. It is also not known whether transcription of thetRNA operon is dependent on p factor. To clarify theseproblems, we have carried out in vitro transcription studieswith the supB-E tRNA genes, using a restriction fragmentfrom the transducing phage (Apsu°2) as template. Structuralanalyses of in vitro transcripts have revealed the sites andsignals for transcription initiation and termination of thetRNA operon.

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In Vitro Transcription of E. coli supB-E tRNA Operon

EXPERIMENTAL PROCEDURES

Materials

Chemicals-Acrylamide and other reagents for gel electrophoresiswere obtained from Wako Pure Chemical Industries, Osaka, Japan.Ribonucleoside 5'-triphosphates (ATP, GTP, CTP, UTP) were ob-tained from P-L Biochemicals, Inc. Urea, ultrapure, was obtainedfrom Schwarz/Mann. DEAE-cellulose plates (PolygramCEL300DEAE) and polyethyleneimine-cellulose plates (PolygramCEL300PEI) were purchased from Machery Nagel Co., Duiren, WestGermany. Yeast RNA was obtained from P-L Biochemicals. E. colitRNA, purchased from Boehringer Mannheim Co., was re-extractedwith phenol, followed by precipitation with ethanol. [8-'4C]ATP (56mCi/mmol), [a-32P]GTP (500 Ci/mmol), and (32 P)orthophosphate(carrier-free) were obtained from New England Nuclear Corp. [5'-32P]pCp' (2000 Ci/mmol) was obtained from Radiochemical Centre,Amersham, U.K. Ribonucleoside y-2P-labeled triphosphates wereprepared as described previously (12).

Enzymes-Ribonucleases T, T2, and U2 were obtained fromSankyo Pharmaceutical Co., Tokyo, Japan. Nuclease P1 was pur-chased from Yamnasa Co., Choshi, Japan. Ribonuclease A and T4RNA ligase were obtained from P-L Biochemicals. DNase I wasobtained from Worthington Biochemical Corp. Polynucleotide kinaseand restriction enzymes were purified as described previously (12).The p factor which was purified as described previously (19) waskindly supplied by Dr. M. Imai of Kyoto University. RNA polymeraseholoenzyme was purified to near homogeneity from E. coli A19according to the method of Sternbach et al. (20) with the followingmodifications. After the step of heparin-Sepharose chromatographyof Sternbach et al. (20), the enzyme fractions were concentrated byammonium sulfate precipitation and dialyzed against 10 mM Tris-HCl(pH 7.8), 10 mM MgCl2, 50 mM KC1, 5 mM 2-mercaptoethanol, 0.1 mmEDTA, 5% glycerol (buffer A). The enzyme was fractionated bySepharose 6B chromatography, followed by DEAE-cellulose chro-matography using a linear gradient between 50 mM KCI/buffer A and300 mM KCl/buffer A. The enzyme preparation was dialyzed against10 mM Tris-HCl (pH 7.8), 50 mm KC1, 0.1 m EDTA, 0.1 mMdithiothreitol, 50% glycerol. The purity of the enzyme preparationwas examined by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (20). The RNA polymerase holoenzyme preparation thuspurified (3 mg of protein/ml) was stored at -20 C.

Methods

Preparation of DNAs-XcI857S7 DNA and psu°2 DNA wereprepared as described previously (12). The Hael.4kb fragment con-taining the supB-E region (12) was prepared as follows. Apsu°2 DNAwas digested with restriction endonucleases Hae III and Eco RI, andthe digests were fractionated by electrophoresis on a 4% polyacryl-amide gel as described previously (12). DNA fragments were visual-ized in the gel after briefly soaking in a solution of 1 pg/ml ethidiumbromide under ultraviolet irradiation. The fragment of 1.4 kilobasepairs was recovered from the gel electrophoretically into dialysis bagsin 50 mM Tris-borate (pH 8.3). The DNA fragment was extractedwith phenol and dialyzed against 10 mM Tris-HCl (pH 7.4), 0.2 mmEDTA, 0.5 M NaCl. After precipitation with ethanol, the DNA frag-ment was dissolved in 10 mM Tris-HCl (pH 7.4), 0.2 mM EDTA.

In Vitro Transcription-Unless indicated otherwise, RNA synthe-sis was carried out in standard reaction mixtures, which contained, in50 pl, 0.2 pmol of template DNA, 4 pl of E. coli RNA polymerase (3mg/ml), 20 mM Tris-HCl (pH 7.8), 10 mm MgCI2, 100 mM KC1, 0.1mm EDTA, 0.1 mM dithiothreitol, and the four ribonucleoside tri-phosphates ATP, GTP, CTP, and UTP (three of which were unla-beled and present in concentrations of 0.1 mm and one of which waslabeled and present in a concentration of 50 pM). The radioactiveprecursors used in various reactions had the following specific activ-ities; [8-'4C]ATP, 20 mCi/mmol; [a-sP]GTP, 100 Ci/mmol; the ribo-nucleoside triphosphates (ATP, GTP, CTP, and UTP) labeled in they positions with 32p, 100 Ci/mmol. Purified p factor (5 g/ml) wasadded where indicated. Transcription reactions were incubated at37 °C for 40 min and terminated by the addition of 200 pl of a solutioncontaining 0.3 M sodium acetate (pH 5.0), 0.2% sodium dodecyl sulfate,and E. coli tRNA (25 pg). The reaction mixture was extracted twice

with phenol and RNA was precipitated with ethanol at -20 C. Afterwashing with 95% ethanol, RNA was dissolved in appropriate buffers.

Polyacrylamide Gel Electrophoresis-Polyacrylamide gel electro-phoresis was carried out between glass plates as described previously(18). Unless indicated otherwise, the size of a standard gel was 20 cmlong x 20 cm wide x 0.1 cm thick. Preparative gels used to isolatelabeled transcription products were 30 cm long x 10 cm wide x 0.2cm thick. The ratio of acrylamide and bisacrylamide was 29:1 and thegel contained 8 M urea. Electrophoresis buffer consisted of 50 mMTris-borate (pH 8.3), 1 mm EDTA. The RNA precipitate was resus-pended in the electrophoresis buffer which contained 8 M urea, 0.01%bromphenol blue dye. The RNA was incubated at 70 °C for 3 minfollowed by quick cooling in ice water and subjected to electropho-resis. Two-dimensional polyacrylamide gel electrophoresis was carriedout without urea as described previously (18). 32 P-labeled RNAs weredetected by autoradiography as described previously (18) and "4C-labeled RNAs were detected by fluorography according to the pro-cedure described by Laskey (21). Elution of RNA from the preparativegels was accomplished by diffusion. The gel, crushed in a 1.5-nmlplastic centrifuge tube, was macerated into 300 Il of a solution of 0.5M sodium acetate (pH 5.0), 1 mM EDTA, 0.2% sodium dodecyl sulfatecontaining 4 pg of E. coli tRNA. Subsequent to incubation at 42 °Cfor 15 h, the acrylamide was removed by centrifugation. The geldebris was re-extracted with 200 pl of the same solution. The RNAwas extracted with phenol and precipitated with ethanol at leastthree times.

Analysis of Terminal Nucleotide Sequences of TranscriptionProducts-For the analysis of 5'-terminal sequences of transcriptionproducts, RNA synthesis was carried out in the reaction mixturecontaining [y- 32 P]GTP. The products were purified by electrophoresison a 4% polyacrylamide gel. Partial nucleotide sequences of the 5'-terminal regions of RNA were determined according to the methoddescribed by Donis-Keller et al. (22).

For the analyses of 3'-terminal sequences of transcription products,RNA synthesis was carried out in the presence of the four unlabeledribonucleoside triphosphates (200 pM each). After incubation at 37 °Cfor 40 min, DNase I (1 ptg) was added to the mixtures and incubationwas continued for an additional 30 min. Subsequent to the additionof 250 p1 of a solution of 0.2% sodium dodecyl sulfate, 0.3 M sodiumacetate (pH 5.0), RNAs were extracted twice with phenol, precipitatedwith ethanol three times, and dried in vacuo. The RNAs wereincubated in reaction mixtures which contained, in 20 pl, 50 mm Tris-HCl (pH 7.5), 15% dimethyl sulfoxide, 10 M MgC12, 5 mM dithio-threitol, 2 pM ATP, 125 pCi of [5'-32 P]pCp (2000 Ci/mmol), and 9units of T4 RNA ligase, at 4 C for 10 h. Ligation reaction wasterminated by the addition of 0.2% sodium dodecyl sulfate, 0.3 Msodium acetate (pH 5.0) (200 p1) containing E. coli tRNA (5 gg). Themixtures were extracted with phenol, and the RNAs were precipitatedwith ethanol. The RNAs were then purified by electrophoresis on 4%polyacrylamide gels. The end-labeled RNA was subjected to partialalkaline hydrolysis according to the protocol described by Donis-Keller et al. (22). The digests were fractionated by two-dimensionalhomochromatography described by Jay et al. (23). Chromatographyfor the second dimension was carried out in the Homo-mix V of Jayet al. (23).

In Vitro Processing of RNA Transcripts-A dialyzed "S30" extractwas prepared from E. coli Q13 as described previously (18). Thetranscripts of the Hael.4kb fragment which were labeled with [a-3 2 P]GTP were electrophoresed on a 5% polyacrylamide gel containing 8M urea, and the RNA of about 800 nucleotides in length was recoveredfrom the gel. The RNA (about 0.1 pmol) was incubated in the reactionmixture containing, in 60 p1, 10 mM Tris-HCI (pH 7.8), 100 mM KCI,0.1 mM 2-mercaptoethanol, 10 pl of the S30 extract (about 180 pg ofprotein) at 37 °C for 2 h. Subsequent to the addition of 250 pl of asolution of 0.2% sodium dodecyl sulfate, 0.3 M sodium acetate (pH5.0) containing E. coli tRNA (5 pg), the RNAs were extracted withphenol, precipitated with ethanol, and analyzed by polyacrylamidegel electrophoresis.

Fingerprint Analysis and Thin Layer Chromatography-AfterRNase T, digestion of 3 2P-labeled RNAs, two-dimensional fingerprintswere made according to the standard procedure (24, 25). Oligonucle-otides were analyzed by RNase T2 digestion, followed by electropho-resis on Whatman 540 paper in pyridine acetate (pH 3.5). Ribonu-cleoside y-s2P-labeled triphosphates derived from the 5' termini ofRNAs by nuclease P1 digestion were analyzed by chromatography ona polyethyleneimine-cellulose plate in 1.5 M KH2 PO 4 (pH 3.4) accord-ing to the procedure described by Cashel et al. (26).

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'The abbreviations used are: pCp, cytidine 5'-3'-diphosphate;Hael.4kb fragment, restriction fragment generated by digestion ofXpsu°2 DNA with Hae III.

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In Vitro Transcription of E. coli supB-E tRNA Operon

GintRNA 1

_ I ALu

A UU-GA-UA-UG'CC'GU G

A

UCU.G

UC UC'GA-UU-A

GintRNA 2

GintRNA 2

MettRNA m

GintRNAG n

FIG. 6. Nucleotide sequence of the transcripts of the supB-EtRNA operon. Mature tRNA sequences are shown schematically incloverleaf form with 3'-terminal tetranucleotides. Other potentialsecondary structures predicted to form in the transcripts are alsoshown. The two heptameric sequences underlined are complemen-

RNase U2, and the digests were electrophoresed on a 20%polyacrylamide gel, we found that the region correspondingto the stem structure was relatively resistant to the nucleases,so that very few digestion products were detected in the gel,although there were six Gs and six As in the region (data notshown). It is likely, therefore, that this region of the transcriptmay form a secondary structure in the reaction mixture, aspredicted from the sequence analysis. The complete nucleo-tide sequence of the transcript of the supB-E tRNA operon isschematically shown in Fig. 6.

DISCUSSION

The present studies have demonstrated that the seventRNA genes in the supB-E region are transcribed in vitro asa single unit from a defined position that is located 33 basepairs upstream from the 5' end of the 5'-proximal tRNA Metgene sequence. The length and nucleotide sequence betweenthe start site and the 5' end of the tRNAme t gene sequence areconsistent with those of the 5' leader sequence of the trimerictRNA precursor (pppG-tRNAXet-tRNALu-tRNA l ) de-tected in the RNase P mutant (18). Thus, in vitro transcrip-tion initiates faithfully at the site where transcription actuallystarts in cells. The transcription start site thus identifiedindicates that the region including the Pribnow box sequence(5'-CATAATG) located 7-13 base pairs upstream from thesite and the sequence in the -35 region represents the pro-moter of the supB-E tRNA operon, as expected from theDNA sequence analysis of the supB-E region (12). In fact,when the -35 region was cleaved with HincII, very littletranscription of the operon was observed. The notion that theseven tRNA genes constitute an operon thus has been verified.Apparently, the multimeric tRNA precursors we observed in

tary with each other and could form stem structure. The arrowsindicate the 3' termini of the transcripts; the largest arrow representsmore than 70% of the chains, whereas the second largest arrowrepresents about 22% of the chains. The smallest arrow indicates the3' termini of about 5% of the major transcripts.

the RNase P mutant upon infection of Apsu°2 must havealready been processed at spacer regions.

As pointed out previously (12), the sequence (CGCCCC)between the Pribnow box sequence and the transcription startsite is identical with that of the tRNAT" gene (4) and verysimilar to those of the tRNA"U genes (29) and the four rRNAoperons (30). The possibility that the G-C-rich sequences inthe region are related to the known stringent control of thegenes was initially suggested with the rRNA operons bydeBoer et al. (30). In fact, it has been demonstrated with thetRNATy gene that the synthetic alteration of the CGCCCCsequence to the CGTTAA sequence alters the in vitro regu-lation of the promoter by ppGpp (31). It remains to be seenwhether in vitro transcription of the supB-E tRNA operon issimilarly regulated by the nucleoside tetraphosphate. In thetRNAThy gene, the eight consecutive GC base pairs located9-16 base pairs upstream from the Pribnow box sequencewere shown to be important for the promoter function (7).Such sequence, however, is not present in the supB-E tRNAoperon, although the presence of three consecutive GC basepairs is noted (12). It has been shown that the tRNATy

promoter is unusually sensitive to the presence of salt, so thatRNA synthesis is virtually abolished in the presence of 100mM KC1 (5). In addition, RNA synthesis is markedly stimu-lated by glycerol at a concentration of 20% (5). The promoterfunction of the supB-E tRNA operon appears not to be sosensitive to the presence of KCI (at concentrations of 50-150mM). Furthermore, in vitro transcription of the supB-E tRNAoperon is relatively unaffected by glycerol in the presence of100 mM KC1. Presumably, the differential effects of salt andglycerol on transcription of the two tRNA operons are relatedto the presence (or absence) of the consecutive GC sequence

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In Vitro Transcription of E. coli supB-E tRNA Operon

in the upstream region of the Pribnow box sequences.The mode of transcription termination of the supB-E tRNA

operon appears to contrast with that of the tRNAT' operon.Whereas transcription termination of the tRNAr operonhas been shown to be p-dependent in vitro as well as in vivo(3, 8), transcription of the supB-E tRNA operon is relativelyunaffected by p factor at least in vitro. Consistent with thep-independent nature of transcription termination of thesupB-E tRNA operon is the occurrence of a potential RNAstem and loop structure immediately before the ends of thetranscripts. Transcription of the supB-E tRNA operon ter-minates at three different sites within the 5'-CTT sequencewhich is located 33-35 base pairs downstream from the 3' endof the second tRNA2"' sequence. The CTT sequence is fol-lowed by two additional Ts and preceded by a sequence of 26nucleotides which could form a hairpin structure of 12 basepairs. This is consistent with the consensus structural featureof p-independent termination sites (17). The region of thepotential stem structure has been shown to be relativelyresistant to RNases T. and U2, suggesting that the region mayform a secondary structure in solution. Among the threetermination sites in the 5'-CTT sequence, the 5'-proximal Tis the preferred position, where more than 70% of the chainsterminate. The C is the second strongest termination point,where about 22% of the chains terminate. Termination at thesecond T accounts for only 5% of the chains. The presence ofnonunique termination sites has also been indicated in thetRNAT" gene (3).

In the transcript of the supB-E tRNA operon, there areother sequences that could form stem and loop structures,including those of amino acid acceptor stems of the tRNAsequences (Fig. 6). Some of the stem structures are followedby relatively U-rich sequences. It is not known whether tran-scription termination occurs, if not efficiently, in these regions.It is possible to assume that the presence of premature ter-mination products in the absence of p factor (Fig. 2) might berelevant to these sequences of the operon. Furthermore, insome spacer regions, there are sequences similar to the 5'-CAATCAA sequence which is present at the p-dependenttermination sites in the tRNATY r gene (2, 3) and the tRI regionof phage X (32). For example, analogous sequences, 5'-CATT-CAC, 5'-CAATCT, 5'-AATTCAA, and 5'-TATTCAA, arepresent in the spacer regions between tRNA "U and tRNA' ",tRNA"'n and tRNA.et, tRNAMet and tRNA2G n", and tRNA2 n'and tRNA2Gn, respectively (Fig. 6). It is worth noting that a 5'-CATTCAA sequence is present 45 base pairs downstreamfrom the second tRNA2' sequence, that is, beyond the p-independent termination sites. It remains to be examinedwhether the slight enhancement of premature termination bythe addition of p factor (Fig. 2) is related, at least in part, tothe presence of some of those sequences in the spacer regions.However, such stimulation of premature termination by pfactor represents a very minor portion of the in vitro tran-scripts and the majority of the transcripts are unaffected bythe presence of p factor.

Transcription termination of tRNA operons, like any othergenes, appears to occur in two different ways; one is p-inde-pendent and the other-is p-dependent. It is not known whichof the two mechanisms is encountered more frequently intranscription of E. coli tRNA genes. Apparently more infor-mation on other tRNA genes is needed to answer the question.

Acknowledgments-We thank Dr. M. Imai for the gift of p factorand Dr. T. Ooi of Kyoto University for performing computer analysisof the secondary structures of nucleotide sequences and for hisvaluable comments.

REFERENCES

1. Ozeki, H. (1980) in RNA-Polymerase, tRNA, and Ribosomes:Their Genetics and Evolution (Osawa, S., Ozeki, H., Uchida,H., and Yura, T., eds) pp. 173-183, Tokyo University Press,Tokyo

2. Egan, J., and Landy, A. (1978) J. Biol. Chem. 253, 3607-36223. Kuiipper, H., Sekiya, T., Rosenberg, M., Egan, J., and Landy, A.

(1978) Nature 272, 423-4284. Sekiya, T., Takeya, T., Contreras, R., Kiipper, H., Khorana, H.

G., and Landy, A. (1976) in RNA Polymerase (Losick, R., andChamberlin, M., eds) pp. 455-472, Cold Spring Harbor Labo-ratory, Cold Spring Harbor, NY

5. Kiipper, H., Contreras, R., Khorana, H. G., and Landy, A. (1976)in RNA Polymerase (Losick, R., and Chamberlin, M., eds) pp.473-484, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY

6. Rossi, J. J., and Landy, A. (1979) Cell 16, 523-5347. Rossi, J. J., Egan, J., Berman, M. L., and Landy, A. (1980) in

RNA Polymerase, tRNA, and Ribosomes: Their Genetics andEvolution (Osawa, S., Ozeki, H., Uchida, H., and Yura, T., eds)pp. 185-208, Tokyo University Press, Tokyo

8. Rossi, J. J., Egan, J., Hudson, L., and Landy, A. (1981) Cell 26,305-314

9. Inokuchi, H., Yamao, F., Sakano, H., and Ozeki, H. (1975) Jpn. J.Genet. 50, 466

10. Inokuchi, H., Yamao, F., Sakano, H., and Ozeki, H. (1979) J. Mol.Biol. 132, 649-662

11. Inokuchi, H., Kodaira, M., Yamao, F., and Ozeki, H. (1979) J.Mol. Biol. 132, 663-677

12. Nakajima, N., Ozeki, H., and Shimura, Y. (1981) Cell 23, 239-24913. Pribnow, D. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 784-78814. Pribnow, D. (1975) J. Mol. Biol. 99, 419-44315. Schaller, H., Gray, C., and Hermann, K. (1975) Proc. Natl. Acad.

Sci. U. S. A. 72, 737-74116. Gilbert, W. (1976) in RNA Polymerase (Losick, R., and Chain-

berlin, M., eds) pp. 193-205, Cold Spring Harbor Laboratory,Cold Spring Harbor, NY

17. Rosenberg, M., and Court, D. (1979) Annu. Rev. Genet. 13,319-353

18. Sakano, H., and Shimura, Y. (1978) J. Mol. Biol. 123, 287-32619. Shigesada, K., and Imai, M. (1978) J. Mol. Biol. 123, 467-48620. Sternbach, H., Engelhardt, R., and Lezius, A. G. (1975) Eur. J.

Biochem. 60, 51-5521. Laskey, R. A. (1980) Methods Enzymol. 65, 363-37122. Donis-Keller, H., Maxam, A. M., and Gilbert, W. (1977) Nucleic

Acids Res. 4, 2527-253823. Jay, E., Bambara, R., Padmanabhan, R., and Wu, R. (1974)

Nucleic Acids Res. 1, 331-35324. Sanger, F., Brownlee, G. G., and Barrell, B. G. (1965) J. Mol. Biol.

13, 373-39825. Barrell, B. G. (1971) in Procedure in Nucleic Acid Research

(Cantoni, G. L., and Davis, D. R., eds) Vol. 2, pp. 751-779,Harper and Row, New York

26. Cashel, M., Lazzarini, R. A., and Kalgacher, B. (1969) J. Chro-matogr. 40, 103-109

27. Rosenberg, M., deCrombrugghe, B., and Weissman, S. (1975) J.Biol. Chem. 250, 4755-4764

28. Roberts, J. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3300-330429. Duester, G., Campen, R. K., and Holmes, W. M. (1981) Nucleic

Acids Res. 9, 2121-213930. deBoer, H. A., Gilbert, S. F., and Nomura, M. (1979) Cell 17,

201-20931. Travers, A. A. (1980) J. Mol. Biol. 141, 91-9732. Rosenberg, M., Court, D., Shimatake, H., Brady, C., and Wulff,

D. L. (1978) Nature 272, 414-423

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N Nakajima, H Ozeki and Y Shimuratranscription products.

In vitro transcription of the supB-E tRNA operon of Escherichia coli. Characterization of

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