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
. 1991 Jul 15;88(14):6018–6022. doi: 10.1073/pnas.88.14.6018

A beta subunit mutation disrupting the catalytic function of Escherichia coli RNA polymerase.

J Lee 1, M Kashlev 1, S Borukhov 1, A Goldfarb 1
PMCID: PMC52013  PMID: 2068078

Abstract

The substitution of the evolutionarily conserved Glu-813 for lysine in the beta subunit of RNA polymerase (RNAP) causes a partial loss of function in the assembled RNAP. In the presence of the four ribonucleoside triphosphates, the mutant RNAP displayed a decreased frequency of promoter clearance and diminished elongation rate. Both defects could be compensated by raising the ribonucleoside triphosphate concentration. In the abortive initiation reaction limited by the incomplete set of ribonucleoside triphosphates, the mutant RNAP generated aberrant patterns of products indicative of their enhanced loss from the RNAP-promoter complex. A model is proposed, attributing the multiple effect of the mutation to the malfunctioning of the RNAP active center.

Full text

PDF
6020

Images in this article

6019Image
on p.6019
6020Image
on p.6020
6020Image
on p.6020
6020Image
on p.6020
6021Image
on p.6021

Selected References

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

  1. Arndt K. M., Chamberlin M. J. RNA chain elongation by Escherichia coli RNA polymerase. Factors affecting the stability of elongating ternary complexes. J Mol Biol. 1990 May 5;213(1):79–108. doi: 10.1016/S0022-2836(05)80123-8. [DOI] [PubMed] [Google Scholar]
  2. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  3. Carpousis A. J., Gralla J. D. Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry. 1980 Jul 8;19(14):3245–3253. doi: 10.1021/bi00555a023. [DOI] [PubMed] [Google Scholar]
  4. Grachev M. A., Lukhtanov E. A., Mustaev A. A., Zaychikov E. F., Abdukayumov M. N., Rabinov I. V., Richter V. I., Skoblov Y. S., Chistyakov P. G. Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling. Eur J Biochem. 1989 Apr 1;180(3):577–585. doi: 10.1111/j.1432-1033.1989.tb14684.x. [DOI] [PubMed] [Google Scholar]
  5. Hansen U. M., McClure W. R. A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase. J Biol Chem. 1979 Jul 10;254(13):5713–5717. [PubMed] [Google Scholar]
  6. Heumann H., Metzger W., Niehörster M. Visualization of intermediary transcription states in the complex between Escherichia coli DNA-dependent RNA polymerases and a promoter-carrying DNA fragment using the gel retardation method. Eur J Biochem. 1986 Aug 1;158(3):575–579. doi: 10.1111/j.1432-1033.1986.tb09793.x. [DOI] [PubMed] [Google Scholar]
  7. Kashlev M., Lee J., Zalenskaya K., Nikiforov V., Goldfarb A. Blocking of the initiation-to-elongation transition by a transdominant RNA polymerase mutation. Science. 1990 May 25;248(4958):1006–1009. doi: 10.1126/science.1693014. [DOI] [PubMed] [Google Scholar]
  8. Kassavetis G. A., Chamberlin M. J. Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro. J Biol Chem. 1981 Mar 25;256(6):2777–2786. [PubMed] [Google Scholar]
  9. Kassavetis G. A., Zentner P. G., Geiduschek E. P. Transcription at bacteriophage T4 variant late promoters. An application of a newly devised promoter-mapping method involving RNA chain retraction. J Biol Chem. 1986 Oct 25;261(30):14256–14265. [PubMed] [Google Scholar]
  10. Krummel B., Chamberlin M. J. RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes. Biochemistry. 1989 Sep 19;28(19):7829–7842. doi: 10.1021/bi00445a045. [DOI] [PubMed] [Google Scholar]
  11. Lee D. N., Phung L., Stewart J., Landick R. Transcription pausing by Escherichia coli RNA polymerase is modulated by downstream DNA sequences. J Biol Chem. 1990 Sep 5;265(25):15145–15153. [PubMed] [Google Scholar]
  12. Lee J. Y., Zalenskaya K., Shin Y. K., McKinney J. D., Park J. H., Goldfarb A. Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase. J Bacteriol. 1989 Jun;171(6):3002–3007. doi: 10.1128/jb.171.6.3002-3007.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Levin J. R., Krummel B., Chamberlin M. J. Isolation and properties of transcribing ternary complexes of Escherichia coli RNA polymerase positioned at a single template base. J Mol Biol. 1987 Jul 5;196(1):85–100. doi: 10.1016/0022-2836(87)90512-2. [DOI] [PubMed] [Google Scholar]
  14. Maizels N. M. The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3585–3589. doi: 10.1073/pnas.70.12.3585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McClure W. R., Cech C. L., Johnston D. E. A steady state assay for the RNA polymerase initiation reaction. J Biol Chem. 1978 Dec 25;253(24):8941–8948. [PubMed] [Google Scholar]
  16. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  17. Metzger W., Schickor P., Heumann H. A cinematographic view of Escherichia coli RNA polymerase translocation. EMBO J. 1989 Sep;8(9):2745–2754. doi: 10.1002/j.1460-2075.1989.tb08416.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Straney D. C., Crothers D. M. A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter. J Mol Biol. 1987 Jan 20;193(2):267–278. doi: 10.1016/0022-2836(87)90218-x. [DOI] [PubMed] [Google Scholar]
  19. Zalenskaya K., Lee J., Gujuluva C. N., Shin Y. K., Slutsky M., Goldfarb A. Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products. Gene. 1990 Apr 30;89(1):7–12. doi: 10.1016/0378-1119(90)90199-2. [DOI] [PubMed] [Google Scholar]
  20. von Hippel P. H., Bear D. G., Morgan W. D., McSwiggen J. A. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem. 1984;53:389–446. doi: 10.1146/annurev.bi.53.070184.002133. [DOI] [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