Abstract
Transcription of the trp operon of Bacillus subtilis is regulated in response to the availability of tryptophan. The first structural gene of the operon is preceded by a 204-base-pair transcribed leader region that contains a segment with the features of a procaryotic termination site. Transcription of the leader region was analyzed in vivo and in vitro to determine whether this putative termination site was used to regulate operon expression. When RNA was isolated from wild-type cells grown in the presence of excess tryptophan, transcripts of the operon ended at the putative termination site. In contrast, RNA isolated from cells grown in the absence of tryptophan or from a mutant strain which is constitutive for trp operon expression contained trp transcripts that extended beyond the termination site into the structural genes. To assess termination quantitatively in vivo, a trpE-lacZ fusion was constructed in which the trp promoter and leader region controls hybrid beta-galactosidase formation. The effects on hybrid beta-galactosidase levels of point mutations and deletions introduced into this leader region were determined. The results obtained establish that transcription of the trp operon structural genes is regulated in the leader region. This regulation appears to be mediated by the formation of alternative secondary structures of the leader transcript. In vitro transcription studies with wild-type and mutant templates provided additional evidence that the identified alternative RNA secondary structures regulate transcription termination. We hypothesize that binding of a tryptophan-activated regulatory protein to a specific segment of the nascent leader transcript prevents formation of one of the alternative secondary structures, thereby directing RNA polymerase to terminate transcription.
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Selected References
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- Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
- Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4174–4178. doi: 10.1073/pnas.73.11.4174. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Band L., Shimotsu H., Henner D. J. Nucleotide sequence of the Bacillus subtilis trpE and trpD genes. Gene. 1984 Jan;27(1):55–65. doi: 10.1016/0378-1119(84)90238-5. [DOI] [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Butler E. T., Chamberlin M. J. Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme. J Biol Chem. 1982 May 25;257(10):5772–5778. [PubMed] [Google Scholar]
- Carlton B. C., Whitt D. D. The isolation and genetic characterization of mutants of the tryptophan system of Bacillus subtilis. Genetics. 1969 Jul;62(3):445–460. doi: 10.1093/genetics/62.3.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
- Clewell D. B., Helinski D. R. Effect of growth conditions on the formation of the relaxation complex of supercoiled ColE1 deoxyribonucleic acid and protein in Escherichia coli. J Bacteriol. 1972 Jun;110(3):1135–1146. doi: 10.1128/jb.110.3.1135-1146.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henner D. J., Band L., Shimotsu H. Nucleotide sequence of the Bacillus subtilis tryptophan operon. Gene. 1985;34(2-3):169–177. doi: 10.1016/0378-1119(85)90125-8. [DOI] [PubMed] [Google Scholar]
- Hoch J. A., Nester E. W. Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis. J Bacteriol. 1973 Oct;116(1):59–66. doi: 10.1128/jb.116.1.59-66.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoch S. O. Mapping of the 5-methyltryptophan resistance locus in Bacillus subtilis. J Bacteriol. 1974 Jan;117(1):315–317. doi: 10.1128/jb.117.1.315-317.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoch S. O., Roth C. W., Crawford I. P., Nester E. W. Control of tryptophan biosynthesis by the methyltryptophan resistance gene in Bacillus subtilis. J Bacteriol. 1971 Jan;105(1):38–45. doi: 10.1128/jb.105.1.38-45.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuroda M. I., Yanofsky C. Evidence for the transcript secondary structures predicted to regulate transcription attenuation in the trp operon. J Biol Chem. 1984 Oct 25;259(20):12838–12843. [PubMed] [Google Scholar]
- Lee F., Yanofsky C. Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4365–4369. doi: 10.1073/pnas.74.10.4365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mott J. E., Galloway J. L., Platt T. Maturation of Escherichia coli tryptophan operon mRNA: evidence for 3' exonucleolytic processing after rho-dependent termination. EMBO J. 1985 Jul;4(7):1887–1891. doi: 10.1002/j.1460-2075.1985.tb03865.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
- Oxender D. L., Zurawski G., Yanofsky C. Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5524–5528. doi: 10.1073/pnas.76.11.5524. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rose J. K., Squires C. L., Yanofsky C., Yang H. L., Zubay G. Regulation of in vitro transcription of the tryptophan operon by purified RNA polymerase in the presence of partially purified repressor and tryptophan. Nat New Biol. 1973 Oct 3;245(144):133–137. doi: 10.1038/newbio245133a0. [DOI] [PubMed] [Google Scholar]
- Rose J. K., Yanofsky C. Interaction of the operator of the tryptophan operon with repressor. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3134–3138. doi: 10.1073/pnas.71.8.3134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
- Rutberg L. Mapping of a temperate bacteriophage active on Bacillus subtilis. J Virol. 1969 Jan;3(1):38–44. doi: 10.1128/jvi.3.1.38-44.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shimotsu H., Henner D. J. Characterization of the Bacillus subtilis tryptophan promoter region. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6315–6319. doi: 10.1073/pnas.81.20.6315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. doi: 10.1073/pnas.44.10.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Squires C. L., Lee F. D., Yanofsky C. Interaction of the trp repressor and RNA polymerase with the trp operon. J Mol Biol. 1975 Feb 15;92(1):93–111. doi: 10.1016/0022-2836(75)90093-5. [DOI] [PubMed] [Google Scholar]
- Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
- Whitt D. D., Carlton B. C. Characterization of mutants with single and multiple defects in the tryptophan biosynthetic pathway in Bacillus subtilis. J Bacteriol. 1968 Oct;96(4):1273–1280. doi: 10.1128/jb.96.4.1273-1280.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang M., Galizzi A., Henner D. Nucleotide sequence of the amylase gene from Bacillus subtilis. Nucleic Acids Res. 1983 Jan 25;11(2):237–249. doi: 10.1093/nar/11.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yanofsky C. Attenuation in the control of expression of bacterial operons. Nature. 1981 Feb 26;289(5800):751–758. doi: 10.1038/289751a0. [DOI] [PubMed] [Google Scholar]
- Yanofsky C. Comparison of regulatory and structural regions of genes of tryptophan metabolism. Mol Biol Evol. 1984 Feb;1(2):143–161. doi: 10.1093/oxfordjournals.molbev.a040307. [DOI] [PubMed] [Google Scholar]
- Yanofsky C., Kelley R. L., Horn V. Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe. J Bacteriol. 1984 Jun;158(3):1018–1024. doi: 10.1128/jb.158.3.1018-1024.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zuber P., Losick R. Use of a lacZ fusion to study the role of the spoO genes of Bacillus subtilis in developmental regulation. Cell. 1983 Nov;35(1):275–283. doi: 10.1016/0092-8674(83)90230-1. [DOI] [PubMed] [Google Scholar]
- Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zurawski G., Elseviers D., Stauffer G. V., Yanofsky C. Translational control of transcription termination at the attenuator of the Escherichia coli tryptophan operon. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5988–5992. doi: 10.1073/pnas.75.12.5988. [DOI] [PMC free article] [PubMed] [Google Scholar]