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. 1980 Oct;77(10):5634–5638. doi: 10.1073/pnas.77.10.5634

Rate-limiting steps in RNA chain initiation.

W R McClure
PMCID: PMC350123  PMID: 6160577

Abstract

Promoter-specific lags in the approach to the steady-state rate of abortive initiation were observed when Escherichia coli RNA polymerase was added to initiate the reaction. The lag times were related to the time required for free enzyme and free promoter to combine and isomerize into a functionally active complex. The lag times measured for several bacteriophage and bacterial promoters differed widely (10 sec to several minutes) and in most cases corresponded to the rate-limiting step in the initiation process. The unique advantage in using the abortive initiation reaction to measure the lags was that the binding and isomerization steps in a simple two-state model could be quantitated separately. The separation of the contributions of both steps was effected by deriving an equation to describe the rate of formation of the active binary complex. Results from experiments based on the theory showed a linear relationship between the observed lag times and the reciprocal enzyme concentration. The slope and intercept of the equation yielded quantitative estimates of the binding and isomerization steps in initiation. The analysis was applied to the bacteriophage T7 A2 and D promoters to show the bases for the differences in in vitro initiation frequency that have been observed for these promoters.

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Selected References

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

  1. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  2. Cech C. L., Lichy J., McClure W. R. Characterization of promoter containing DNA fragments based on the abortive initiation reaction of Escherichia coli RNA polymerase. J Biol Chem. 1980 Mar 10;255(5):1763–1766. [PubMed] [Google Scholar]
  3. Cech C. L., McClure W. R. Characterization of ribonucleic acid polymerase-T7 promoter binary complexes. Biochemistry. 1980 May 27;19(11):2440–2447. doi: 10.1021/bi00552a023. [DOI] [PubMed] [Google Scholar]
  4. Chamberlin M. J. The selectivity of transcription. Annu Rev Biochem. 1974;43(0):721–775. doi: 10.1146/annurev.bi.43.070174.003445. [DOI] [PubMed] [Google Scholar]
  5. Darlix J. L., Fromageot P. Discontinuous in vitro transcription of DNA. Biochimie. 1972;54(1):47–54. doi: 10.1016/s0300-9084(72)80037-3. [DOI] [PubMed] [Google Scholar]
  6. DiLauro R., Taniguchi T., Musso R., de Crombrugghe B. Unusual location and function of the operator in the Escherichia coli galactose operon. Nature. 1979 Jun 7;279(5713):494–500. doi: 10.1038/279494a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Johnsrud L. Contacts between Escherichia coli RNA polymerase and a lac operon promoter. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5314–5318. doi: 10.1073/pnas.75.11.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kennell D., Riezman H. Transcription and translation initiation frequencies of the Escherichia coli lac operon. J Mol Biol. 1977 Jul;114(1):1–21. doi: 10.1016/0022-2836(77)90279-0. [DOI] [PubMed] [Google Scholar]
  10. Lowe P. A., Hager D. A., Burgess R. R. Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry. 1979 Apr 3;18(7):1344–1352. doi: 10.1021/bi00574a034. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Maquat L. E., Reznikoff W. S. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters. J Mol Biol. 1978 Nov 15;125(4):467–490. doi: 10.1016/0022-2836(78)90311-x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Nierman W. C., Chamberlin M. J. Studies of RNA chain initiation by Escherichia coli RNA polymerase bound to T7 DNA. Direct analysis of the kinetics and extent of RNA chain initiation at T7 promoter A1. J Biol Chem. 1979 Aug 25;254(16):7921–7926. [PubMed] [Google Scholar]
  15. Pace N. R. Structure and synthesis of the ribosomal ribonucleic acid of prokaryotes. Bacteriol Rev. 1973 Dec;37(4):562–603. doi: 10.1128/br.37.4.562-603.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Seeburg P. H., Nüsslein C., Schaller H. Interaction of RNA polymerase with promoters from bacteriophage fd. Eur J Biochem. 1977 Mar 15;74(1):107–113. doi: 10.1111/j.1432-1033.1977.tb11372.x. [DOI] [PubMed] [Google Scholar]
  18. Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
  19. Stahl S. J., Chamberlin M. J. An expanded transcriptional map of T7 bacteriophage. Reading of minor T7 promoter sites in vitro by Escherichia coli RNA polymerase. J Mol Biol. 1977 Jun 5;112(4):577–601. doi: 10.1016/s0022-2836(77)80165-4. [DOI] [PubMed] [Google Scholar]
  20. Stefano J. E., Gralla J. Lac UV5 transcription in vitro. Rate limitation subsequent to formation of an RNA polymerase-DNA complex. Biochemistry. 1979 Mar 20;18(6):1063–1067. doi: 10.1021/bi00573a020. [DOI] [PubMed] [Google Scholar]
  21. Strickland S., Palmer G., Massey V. Determination of dissociation constants and specific rate constants of enzyme-substrate (or protein-ligand) interactions from rapid reaction kinetic data. J Biol Chem. 1975 Jun 10;250(11):4048–4052. [PubMed] [Google Scholar]
  22. Walter G., Zillig W., Palm P., Fuchs E. Initiation of DNA-dependent RNA synthesis and the effect of heparin on RNA polymerase. Eur J Biochem. 1967 Dec;3(2):194–201. doi: 10.1111/j.1432-1033.1967.tb19515.x. [DOI] [PubMed] [Google Scholar]
  23. von Gabain A., Bujard H. Interaction of Escherichia coli RNA polymerase with promoters of several coliphage and plasmid DNAs. Proc Natl Acad Sci U S A. 1979 Jan;76(1):189–193. doi: 10.1073/pnas.76.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]

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