Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Sep 25;20(18):4773–4779. doi: 10.1093/nar/20.18.4773

Transcription frequency modulates the efficiency of an attenuator preceding the rpoBC RNA polymerase genes of Escherichia coli: possible autogenous control.

K L Steward 1, T Linn 1
PMCID: PMC334231  PMID: 1408790

Abstract

Expression of the rpoBC genes encoding the beta and beta' RNA polymerase subunits of Escherichia coli is autogenously regulated. Although previous studies have demonstrated a post-transcriptional feedback mechanism, complex transcriptional controls of rpoBC expression may also contribute. We show that an attenuator (rpoBa) separating the ribosomal protein (rpl) genes from the rpoBC genes in the rplKAJLrpoBC gene cluster is modulated in its efficiency in response to changes in the frequency of transcription initiated by promoters located upstream. A series of rplJLrpoBalacZ transcriptional fusions was constructed on lambda vectors in which transcription into the rpoBa attenuator was varied by using a variety of promoters with different strengths. beta-galactosidase assays performed on monolysogens of the recombinant phage show that with transcription increasing over a 40-fold range, readthrough of rpoBa decreases from 61% to 19%. In contrast, two other well-characterized terminators show nearly constant efficiencies over a similar range of transcription frequencies. Using a set of phage P22 ant promoter variants with single-nucleotide changes in the promoter consensus sequences also demonstrates that the modulation of rpoBa function appears to be unrelated to the phenomenon of 'factor-independent antitermination' reported by others. The implications for autogenous control of RNA polymerase synthesis are discussed.

Full text

PDF
4773

Selected References

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

  1. Albrechtsen B., Squires C. L., Li S., Squires C. Antitermination of characterized transcriptional terminators by the Escherichia coli rrnG leader region. J Mol Biol. 1990 May 5;213(1):123–134. doi: 10.1016/S0022-2836(05)80125-1. [DOI] [PubMed] [Google Scholar]
  2. Almond N., Yajnik V., Svec P., Godson G. N. An Escherichia coli cis-acting antiterminator sequence: the dnaG nut site. Mol Gen Genet. 1989 Apr;216(2-3):195–203. doi: 10.1007/BF00334356. [DOI] [PubMed] [Google Scholar]
  3. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  4. Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barry G., Squires C. L., Squires C. Control features within the rplJL-rpoBC transcription unit of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4922–4926. doi: 10.1073/pnas.76.10.4922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barry G., Squires C., Squires C. L. Attenuation and processing of RNA from the rplJL--rpoBC transcription unit of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3331–3335. doi: 10.1073/pnas.77.6.3331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blumenthal R. M., Dennis P. P. Regulation of ribonucleic acid polymerase synthesis during restriction of an Escherichia coli mutant temperature sensitive for transcription factor sigma. J Bacteriol. 1980 Jun;142(3):1049–1054. doi: 10.1128/jb.142.3.1049-1054.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  9. Brosius J., Holy A. Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6929–6933. doi: 10.1073/pnas.81.22.6929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cromie K. D., Hayward R. S. Evidence for rifampicin-promoted readthrough of a fully rho-dependent transcriptional terminator. Mol Gen Genet. 1984;193(3):532–534. doi: 10.1007/BF00382095. [DOI] [PubMed] [Google Scholar]
  11. Dennis P. P., Nene V., Glass R. E. Autogenous posttranscriptional regulation of RNA polymerase beta and beta' subunit synthesis in Escherichia coli. J Bacteriol. 1985 Feb;161(2):803–806. doi: 10.1128/jb.161.2.803-806.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Downing W. L., Dennis P. P. Transcription products from the rplKAJL-rpoBC gene cluster. J Mol Biol. 1987 Apr 20;194(4):609–620. doi: 10.1016/0022-2836(87)90238-5. [DOI] [PubMed] [Google Scholar]
  13. Downing W., Dennis P. P. RNA polymerase activity may regulate transcription initiation and attenuation in the rplKAJLrpoBC operon in Escherichia coli. J Biol Chem. 1991 Jan 15;266(2):1304–1311. [PubMed] [Google Scholar]
  14. Drlica K., Franco R. J., Steck T. R. Rifampin and rpoB mutations can alter DNA supercoiling in Escherichia coli. J Bacteriol. 1988 Oct;170(10):4983–4985. doi: 10.1128/jb.170.10.4983-4985.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Farnham P. J., Greenblatt J., Platt T. Effects of NusA protein on transcription termination in the tryptophan operon of Escherichia coli. Cell. 1982 Jul;29(3):945–951. doi: 10.1016/0092-8674(82)90457-3. [DOI] [PubMed] [Google Scholar]
  16. Fukuda R., Nagasawa-Fujimori H. Mechanism of the rifampicin induction of RNA polymerase beta and beta' subunit synthesis in Escherichia coli. J Biol Chem. 1983 Feb 25;258(4):2720–2728. [PubMed] [Google Scholar]
  17. Fukuda R., Taketo M., Ishihama A. Autogenous regulation of RNA polymerase beta subunit synthesis in vitro. J Biol Chem. 1978 Jul 10;253(13):4501–4504. [PubMed] [Google Scholar]
  18. Goliger J. A., Yang X. J., Guo H. C., Roberts J. W. Early transcribed sequences affect termination efficiency of Escherichia coli RNA polymerase. J Mol Biol. 1989 Jan 20;205(2):331–341. doi: 10.1016/0022-2836(89)90344-6. [DOI] [PubMed] [Google Scholar]
  19. Greenblatt J., McLimont M., Hanly S. Termination of transcription by nusA gene protein of Escherichia coli. Nature. 1981 Jul 16;292(5820):215–220. doi: 10.1038/292215a0. [DOI] [PubMed] [Google Scholar]
  20. Hayward R. S., Fyfe S. Over-synthesis and instability of sigma protein in a merodiploid strain of Escherichia coli. Mol Gen Genet. 1978 Feb 7;159(1):89–99. doi: 10.1007/BF00401752. [DOI] [PubMed] [Google Scholar]
  21. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  22. Iwakura Y., Ito K., Ishihama A. Biosynthesis of RNA polymerase in Escherichia coli. I. Control of RNA polymerase content at various growth rates. Mol Gen Genet. 1974;133(1):1–23. doi: 10.1007/BF00268673. [DOI] [PubMed] [Google Scholar]
  23. Kajitani M., Fukuda R., Ishihama A. Autogenous and post-transcriptional regulation of Escherichia coli RNA polymerase synthesis in vitro. Mol Gen Genet. 1980;179(3):489–496. doi: 10.1007/BF00271738. [DOI] [PubMed] [Google Scholar]
  24. Kawakami K., Saitoh T., Ishihama A. Biosynthesis of RNA polymerase in Escherichia coli. IX. Growth-dependent variations in the synthesis rate, content and distribution of RNA polymerase. Mol Gen Genet. 1979 Jul 13;174(2):107–116. doi: 10.1007/BF00268348. [DOI] [PubMed] [Google Scholar]
  25. Lau L. F., Roberts J. W., Wu R. RNA polymerase pausing and transcript release at the lambda tR1 terminator in vitro. J Biol Chem. 1983 Aug 10;258(15):9391–9397. [PubMed] [Google Scholar]
  26. Li S. C., Squires C. L., Squires C. Antitermination of E. coli rRNA transcription is caused by a control region segment containing lambda nut-like sequences. Cell. 1984 Oct;38(3):851–860. doi: 10.1016/0092-8674(84)90280-0. [DOI] [PubMed] [Google Scholar]
  27. Linn T., Greenblatt J. The NusA and NusG proteins of Escherichia coli increase the in vitro readthrough frequency of a transcriptional attenuator preceding the gene for the beta subunit of RNA polymerase. J Biol Chem. 1992 Jan 25;267(3):1449–1454. [PubMed] [Google Scholar]
  28. Linn T., Ralling G. A versatile multiple- and single-copy vector system for the in vitro construction of transcriptional fusions to lacZ. Plasmid. 1985 Sep;14(2):134–142. doi: 10.1016/0147-619x(85)90073-3. [DOI] [PubMed] [Google Scholar]
  29. Linn T., St Pierre R. Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ. J Bacteriol. 1990 Feb;172(2):1077–1084. doi: 10.1128/jb.172.2.1077-1084.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Little R., Dennis P. P. Regulation of RNA polymerase synthesis. Conditional lethal amber mutations in the beta subunit gene. J Biol Chem. 1980 Apr 25;255(8):3536–3541. [PubMed] [Google Scholar]
  31. Mason S. W., Greenblatt J. Assembly of transcription elongation complexes containing the N protein of phage lambda and the Escherichia coli elongation factors NusA, NusB, NusG, and S10. Genes Dev. 1991 Aug;5(8):1504–1512. doi: 10.1101/gad.5.8.1504. [DOI] [PubMed] [Google Scholar]
  32. Meek D. W., Hayward R. S. Direct evidence for autogenous regulation of the Escherichia coli genes rpoBC in vivo. Mol Gen Genet. 1986 Mar;202(3):500–508. doi: 10.1007/BF00333284. [DOI] [PubMed] [Google Scholar]
  33. Morgan B. A., Hayward R. S. Direct evidence for rifampicin-promoted readthrough of the partial terminator tL7 in the rpoBC operon of Escherichia coli. Mol Gen Genet. 1987 Dec;210(2):358–363. doi: 10.1007/BF00325706. [DOI] [PubMed] [Google Scholar]
  34. Morgan E. A. Antitermination mechanisms in rRNA operons of Escherichia coli. J Bacteriol. 1986 Oct;168(1):1–5. doi: 10.1128/jb.168.1.1-5.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mousset S., Thomas R. Ter, a function which generates the ends of the mature lambda chromosome. Nature. 1969 Jan 18;221(5177):242–244. doi: 10.1038/221242a0. [DOI] [PubMed] [Google Scholar]
  36. Moyle H., Waldburger C., Susskind M. M. Hierarchies of base pair preferences in the P22 ant promoter. J Bacteriol. 1991 Mar;173(6):1944–1950. doi: 10.1128/jb.173.6.1944-1950.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Newman A. J., Ma J. C., Howe K. M., Garner I., Hayward R. S. Evidence that rifampicin can stimulate readthrough of transcriptional terminators in Escherichia coli, including the attenuator of the rpoBC operon. Nucleic Acids Res. 1982 Nov 25;10(22):7409–7424. doi: 10.1093/nar/10.22.7409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nodwell J. R., Greenblatt J. The nut site of bacteriophage lambda is made of RNA and is bound by transcription antitermination factors on the surface of RNA polymerase. Genes Dev. 1991 Nov;5(11):2141–2151. doi: 10.1101/gad.5.11.2141. [DOI] [PubMed] [Google Scholar]
  39. Passador L., Linn T. Autogenous regulation of the RNA polymerase beta subunit of Escherichia coli occurs at the translational level in vivo. J Bacteriol. 1989 Nov;171(11):6234–6242. doi: 10.1128/jb.171.11.6234-6242.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Peacock S., Cenatiempo Y., Robakis N., Brot N., Weissbach H. In vitro synthesis of the first dipeptide of the beta subunit of Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4609–4612. doi: 10.1073/pnas.79.15.4609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pedersen S., Reeh S. V. Analysis of the proteins synthesized in ultraviolet light-irradiated Escherichia coli following infection with the bacteriophages lambdadrifd 18 and lambdadfus-3. Mol Gen Genet. 1976 Mar 30;144(3):339–343. doi: 10.1007/BF00341733. [DOI] [PubMed] [Google Scholar]
  42. Post L. E., Strycharz G. D., Nomura M., Lewis H., Dennis P. P. Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1697–1701. doi: 10.1073/pnas.76.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pruss G. J., Drlica K. DNA supercoiling and prokaryotic transcription. Cell. 1989 Feb 24;56(4):521–523. doi: 10.1016/0092-8674(89)90574-6. [DOI] [PubMed] [Google Scholar]
  44. Ralling G., Bodrug S., Linn T. Growth rate-dependent regulation of RNA polymerase synthesis in Escherichia coli. Mol Gen Genet. 1985;201(3):379–386. doi: 10.1007/BF00331327. [DOI] [PubMed] [Google Scholar]
  45. Ralling G., Linn T. Evidence that Rho and NusA are involved in termination in the rplL-rpoB intercistronic region. J Bacteriol. 1987 May;169(5):2277–2280. doi: 10.1128/jb.169.5.2277-2280.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ralling G., Linn T. Relative activities of the transcriptional regulatory sites in the rplKAJLrpoBC gene cluster of Escherichia coli. J Bacteriol. 1984 Apr;158(1):279–285. doi: 10.1128/jb.158.1.279-285.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Santos M. A. An improved method for the small scale preparation of bacteriophage DNA based on phage precipitation by zinc chloride. Nucleic Acids Res. 1991 Oct 11;19(19):5442–5442. doi: 10.1093/nar/19.19.5442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schmidt M. C., Chamberlin M. J. nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites. J Mol Biol. 1987 Jun 20;195(4):809–818. doi: 10.1016/0022-2836(87)90486-4. [DOI] [PubMed] [Google Scholar]
  49. Schmidt M. C., Chamberlin M. J. nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites. J Mol Biol. 1987 Jun 20;195(4):809–818. doi: 10.1016/0022-2836(87)90486-4. [DOI] [PubMed] [Google Scholar]
  50. Sharrock R. A., Gourse R. L., Nomura M. Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5275–5279. doi: 10.1073/pnas.82.16.5275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Steward K. L., Linn T. In vivo analysis of overlapping transcription units in the rplKAJLrpoBC ribosomal protein-RNA polymerase gene cluster of Escherichia coli. J Mol Biol. 1991 Mar 5;218(1):23–31. doi: 10.1016/0022-2836(91)90870-c. [DOI] [PubMed] [Google Scholar]
  52. Telesnitsky A. P., Chamberlin M. J. Sequences linked to prokaryotic promoters can affect the efficiency of downstream termination sites. J Mol Biol. 1989 Jan 20;205(2):315–330. doi: 10.1016/0022-2836(89)90343-4. [DOI] [PubMed] [Google Scholar]
  53. Ward D. F., Gottesman M. E. The nus mutations affect transcription termination in Escherichia coli. Nature. 1981 Jul 16;292(5820):212–215. doi: 10.1038/292212a0. [DOI] [PubMed] [Google Scholar]
  54. Whalen W., Ghosh B., Das A. NusA protein is necessary and sufficient in vitro for phage lambda N gene product to suppress a rho-independent terminator placed downstream of nutL. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2494–2498. doi: 10.1073/pnas.85.8.2494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yager T. D., von Hippel P. H. A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli. Biochemistry. 1991 Jan 29;30(4):1097–1118. doi: 10.1021/bi00218a032. [DOI] [PubMed] [Google Scholar]
  56. von Hippel P. H., Yager T. D. Transcript elongation and termination are competitive kinetic processes. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2307–2311. doi: 10.1073/pnas.88.6.2307. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES