Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Oct 1980; 77(10): 5634–5638.
PMCID: PMC350123

Rate-limiting steps in RNA chain initiation.


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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Rosenberg M, Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. [PubMed]
  • Siebenlist U, Simpson RB, Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. [PubMed]
  • 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. [PubMed]
  • Chamberlin MJ. The selectivity of transcription. Annu Rev Biochem. 1974;43(0):721–775. [PubMed]
  • Nierman WC, Chamberlin MJ. 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]
  • Seeburg PH, Nüsslein C, Schaller H. Interaction of RNA polymerase with promoters from bacteriophage fd. Eur J Biochem. 1977 Mar 15;74(1):107–113. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Stahl SJ, Chamberlin MJ. 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. [PubMed]
  • Maquat LE, Reznikoff WS. 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. [PubMed]
  • McClure WR, Cech CL, Johnston DE. A steady state assay for the RNA polymerase initiation reaction. J Biol Chem. 1978 Dec 25;253(24):8941–8948. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Burgess RR, Jendrisak JJ. 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. [PubMed]
  • Lowe PA, Hager DA, Burgess RR. Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry. 1979 Apr 3;18(7):1344–1352. [PubMed]
  • Cech CL, Lichy J, McClure WR. 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]
  • 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]
  • Cech CL, McClure WR. Characterization of ribonucleic acid polymerase-T7 promoter binary complexes. Biochemistry. 1980 May 27;19(11):2440–2447. [PubMed]
  • Hansen UM, McClure WR. A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase. J Biol Chem. 1979 Jul 10;254(13):5713–5717. [PubMed]
  • Maizels NM. 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. [PMC free article] [PubMed]
  • Darlix JL, Fromageot P. Discontinuous in vitro transcription of DNA. Biochimie. 1972;54(1):47–54. [PubMed]
  • 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. [PubMed]
  • Stefano JE, 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. [PubMed]
  • Pace NR. Structure and synthesis of the ribosomal ribonucleic acid of prokaryotes. Bacteriol Rev. 1973 Dec;37(4):562–603. [PMC free article] [PubMed]
  • Kennell D, Riezman H. Transcription and translation initiation frequencies of the Escherichia coli lac operon. J Mol Biol. 1977 Jul;114(1):1–21. [PubMed]

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


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...