Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1977 Nov;4(11):3863–3876. doi: 10.1093/nar/4.11.3863

Steady state kinetic studies of initiation of RNA synthesis on T7 DNA in the presence of rifampicin.

J W Smagowicz, K H Scheit
PMCID: PMC343206  PMID: 593891

Abstract

The steady state kinetics of initiation of T7 DNA transcription by RNA polymerase holo enzyme from E. coli in the presence of rifampicin and the two substrates ATP and UTP were studied. Under these conditions, the enzyme catalyzes exclusively the promotor specific synthesis of pppApU. The kinetic data are in agreement with the mechanism of a truly ordered reaction. Binding of the initiating nucleotide ATP to the transcriptional complex occurs prior to the binding of the substrate UTP. Release of pppApU is most probably the rate limitinig step. Km constants were found to be 0.6 mM for ATP and 0.31 mM for UTP, respectively. The substrate inhibition pattern indicated that the substrate site exhibits a finite affinity for incorrect nucleoside triphosphate (Ki = 2.3 mM). A similar non specific binding to the 3-OH site could not be demonstrated.

Full text

PDF
3863

Selected References

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

  1. Downey K. M., So A. G. Studies on the kinetics of ribonucleic acid chain initiation and elongation. Biochemistry. 1970 Jun 9;9(12):2520–2525. doi: 10.1021/bi00814a019. [DOI] [PubMed] [Google Scholar]
  2. Lowery C., Richardson J. P. Characterization of the nucleoside triphosphate phosphohydrolase (ATPase) activity of RNA synthesi termination factor p. I. Enzymatic properties and effects of inhibitors. J Biol Chem. 1977 Feb 25;252(4):1375–1380. [PubMed] [Google Scholar]
  3. Mangel W. F., Chamberlin M. J. Studies of ribonucleic acid chain initiation by Escherichia coli ribonucleic acid polymerase bound to T7 deoxyribonucleic acid. I. An assay for the rate and extent of ribonucleic acid chain initiation. J Biol Chem. 1974 May 25;249(10):2995–3001. [PubMed] [Google Scholar]
  4. Rhodes G., Chamberlin M. J. Kinetic analysis of ribonucleic acid chain initiation by Escherichia coli Ribonucleic acid polymerase bound to DNA. J Biol Chem. 1975 Dec 10;250(23):9112–9120. [PubMed] [Google Scholar]
  5. Rhodes G., Chamberlin M. J. Ribonucleic acid chain elongation by Escherichia coli ribonucleic acid polymerase. I. Isolation of ternary complexes and the kinetics of elongation. J Biol Chem. 1974 Oct 25;249(20):6675–6683. [PubMed] [Google Scholar]
  6. Sternbach H., Engelhardt R., Lezius A. G. Rapid isolation of highly active RNA polymerase from Escherichia coli and its subunits by matrix-bound heparin. Eur J Biochem. 1975 Dec 1;60(1):51–55. doi: 10.1111/j.1432-1033.1975.tb20974.x. [DOI] [PubMed] [Google Scholar]
  7. Terao T., Dahlberg J. E., Khorana H. G. Studies on polynucleotides. CXX. On the transcription of a synthetic 29-unit long deoxyribopolynucleotide. J Biol Chem. 1972 Oct 10;247(19):6157–6166. [PubMed] [Google Scholar]
  8. Zillig W., Zechel K., Halbwachs H. J. A new method of large scale preparation of highly purified DNA-dependent RNA-polymerase from E. coli. Hoppe Seylers Z Physiol Chem. 1970 Feb;351(2):221–224. doi: 10.1515/bchm2.1970.351.1.221. [DOI] [PubMed] [Google Scholar]

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

RESOURCES