Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Jan 1;90(1):133–137. doi: 10.1073/pnas.90.1.133

Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein.

P Babitzke 1, C Yanofsky 1
PMCID: PMC45614  PMID: 7678334

Abstract

We have reconstituted Bacillus subtilis trp attenuation in vitro. Purification of the mtrB gene product (TRAP) to near homogeneity allowed us to demonstrate that addition of this protein plus L-tryptophan to template, RNA polymerase, and nucleoside triphosphates caused transcription termination in the trpEDCFBA leader region. TRAP acts by binding to the nascent transcript and preventing formation of an RNA antiterminator structure, thereby allowing terminator formation and transcription termination. Oligonucleotides complementary to segments of the antiterminator were used to demonstrate that formation of this RNA hairpin was responsible for transcription read-through. TRAP was found to be a 60-kDa multimeric protein composed of identical 6- to 8-kDa subunits, and its elution profile on a chromatographic column did not change in the presence of tryptophan.

Full text

PDF
137

Images in this article

134Image
on p.134
135Image
on p.135
135Image
on p.135
136Image
on p.136

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amster-Choder O., Houman F., Wright A. Protein phosphorylation regulates transcription of the beta-glucoside utilization operon in E. coli. Cell. 1989 Sep 8;58(5):847–855. doi: 10.1016/0092-8674(89)90937-9. [DOI] [PubMed] [Google Scholar]
  2. Babitzke P., Gollnick P., Yanofsky C. The mtrAB operon of Bacillus subtilis encodes GTP cyclohydrolase I (MtrA), an enzyme involved in folic acid biosynthesis, and MtrB, a regulator of tryptophan biosynthesis. J Bacteriol. 1992 Apr;174(7):2059–2064. doi: 10.1128/jb.174.7.2059-2064.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  5. Debarbouille M., Arnaud M., Fouet A., Klier A., Rapoport G. The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators. J Bacteriol. 1990 Jul;172(7):3966–3973. doi: 10.1128/jb.172.7.3966-3973.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  7. Gollnick P., Ishino S., Kuroda M. I., Henner D. J., Yanofsky C. The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8726–8730. doi: 10.1073/pnas.87.22.8726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Holmberg C., Rutberg B. Expression of the gene encoding glycerol-3-phosphate dehydrogenase (glpD) in Bacillus subtilis is controlled by antitermination. Mol Microbiol. 1991 Dec;5(12):2891–2900. doi: 10.1111/j.1365-2958.1991.tb01849.x. [DOI] [PubMed] [Google Scholar]
  10. Houman F., Diaz-Torres M. R., Wright A. Transcriptional antitermination in the bgl operon of E. coli is modulated by a specific RNA binding protein. Cell. 1990 Sep 21;62(6):1153–1163. doi: 10.1016/0092-8674(90)90392-r. [DOI] [PubMed] [Google Scholar]
  11. Jozwik C. E., Miller E. S. Regions of bacteriophage T4 and RB69 RegA translational repressor proteins that determine RNA-binding specificity. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5053–5057. doi: 10.1073/pnas.89.11.5053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kuroda M. I., Henner D., Yanofsky C. cis-acting sites in the transcript of the Bacillus subtilis trp operon regulate expression of the operon. J Bacteriol. 1988 Jul;170(7):3080–3088. doi: 10.1128/jb.170.7.3080-3088.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kuroda M. I., Shimotsu H., Henner D. J., Yanofsky C. Regulatory elements common to the Bacillus pumilus and Bacillus subtilis trp operons. J Bacteriol. 1986 Sep;167(3):792–798. doi: 10.1128/jb.167.3.792-798.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Martin-Verstraete I., Débarbouillé M., Klier A., Rapoport G. Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon. J Mol Biol. 1990 Aug 5;214(3):657–671. doi: 10.1016/0022-2836(90)90284-S. [DOI] [PubMed] [Google Scholar]
  16. Murphy N., McConnell D. J., Cantwell B. A. The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme beta-glucanase. Nucleic Acids Res. 1984 Jul 11;12(13):5355–5367. doi: 10.1093/nar/12.13.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Otridge J., Gollnick P. MtrB from Bacillus subtilis binds specifically to trp leader RNA in a tryptophan-dependent manner. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):128–132. doi: 10.1073/pnas.90.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Otwinowski Z., Schevitz R. W., Zhang R. G., Lawson C. L., Joachimiak A., Marmorstein R. Q., Luisi B. F., Sigler P. B. Crystal structure of trp repressor/operator complex at atomic resolution. Nature. 1988 Sep 22;335(6188):321–329. doi: 10.1038/335321a0. [DOI] [PubMed] [Google Scholar]
  19. Schevitz R. W., Otwinowski Z., Joachimiak A., Lawson C. L., Sigler P. B. The three-dimensional structure of trp repressor. 1985 Oct 31-Nov 6Nature. 317(6040):782–786. doi: 10.1038/317782a0. [DOI] [PubMed] [Google Scholar]
  20. Schnetz K., Toloczyki C., Rak B. Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes. J Bacteriol. 1987 Jun;169(6):2579–2590. doi: 10.1128/jb.169.6.2579-2590.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sheline C. T., Milocco L. H., Jones K. A. Two distinct nuclear transcription factors recognize loop and bulge residues of the HIV-1 TAR RNA hairpin. Genes Dev. 1991 Dec;5(12B):2508–2520. doi: 10.1101/gad.5.12b.2508. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Shimotsu H., Henner D. J. Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes. J Bacteriol. 1986 Oct;168(1):380–388. doi: 10.1128/jb.168.1.380-388.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shimotsu H., Kuroda M. I., Yanofsky C., Henner D. J. Novel form of transcription attenuation regulates expression the Bacillus subtilis tryptophan operon. J Bacteriol. 1986 May;166(2):461–471. doi: 10.1128/jb.166.2.461-471.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Whalen W. A., Das A. Action of an RNA site at a distance: role of the nut genetic signal in transcription antitermination by phage-lambda N gene product. New Biol. 1990 Nov;2(11):975–991. [PubMed] [Google Scholar]
  27. Wilson S., Drew R. Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product. J Bacteriol. 1991 Aug;173(16):4914–4921. doi: 10.1128/jb.173.16.4914-4921.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yanofsky C. Transcription attenuation. J Biol Chem. 1988 Jan 15;263(2):609–612. [PubMed] [Google Scholar]
  29. Zalkin H., Ebbole D. J. Organization and regulation of genes encoding biosynthetic enzymes in Bacillus subtilis. J Biol Chem. 1988 Feb 5;263(4):1595–1598. [PubMed] [Google Scholar]
  30. el Hassouni M., Henrissat B., Chippaux M., Barras F. Nucleotide sequences of the arb genes, which control beta-glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new beta-glycohydrolase family including enzymes from eubacteria, archeabacteria, and humans. J Bacteriol. 1992 Feb;174(3):765–777. doi: 10.1128/jb.174.3.765-777.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES