Visualization and Quantitative Analysis of Complex Formation Between E. Coli RNA Polymerase and an RRNA Promoter in Vitro

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1988 Oct 25

PMID 3054811

Citations 53

Authors

R L Gourse

Affiliations

Soon will be listed here.

Abstract

We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotide production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polymerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.

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References

Peacock A, DINGMAN C . Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry. 1968; 7(2):668-74. DOI: 10.1021/bi00842a023. View

Szoke P, Allen T, deHaseth P . Promoter recognition by Escherichia coli RNA polymerase: effects of base substitutions in the -10 and -35 regions. Biochemistry. 1987; 26(19):6188-94. DOI: 10.1021/bi00393a035. View

McClure W, Cech C, Johnston D . A steady state assay for the RNA polymerase initiation reaction. J Biol Chem. 1978; 253(24):8941-8. View

Glaser G, CASHEL M . In vitro transcripts from the rrn B ribosomal RNA cistron originate from two tandem promoters. Cell. 1979; 16(1):111-21. DOI: 10.1016/0092-8674(79)90192-2. View

Gilbert S, DE BOER H, Nomura M . Identification of initiation sites for the in vitro transcription of rRNA operons rrnE and rrnA in E. coli. Cell. 1979; 17(1):211-24. DOI: 10.1016/0092-8674(79)90309-x. View

Hamming J, Gruber M, Ab G . Interaction between RNA polymerase and a ribosomal RNA promoter of E. coli. Nucleic Acids Res. 1979; 7(4):1019-33. PMC: 342279. DOI: 10.1093/nar/7.4.1019. View

Gallant J . Stringent control in E. coli. Annu Rev Genet. 1979; 13:393-415. DOI: 10.1146/annurev.ge.13.120179.002141. View

Maxam A, Gilbert W . Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980; 65(1):499-560. DOI: 10.1016/s0076-6879(80)65059-9. View

Carpousis A, Gralla J . Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry. 1980; 19(14):3245-53. DOI: 10.1021/bi00555a023. View

10.

Hamming J, Ab G, Gruber M . E coli RNA polymerase-rRNA promoter interaction and the effect of ppGpp. Nucleic Acids Res. 1980; 8(17):3947-63. PMC: 324206. DOI: 10.1093/nar/8.17.3947. View

11.

McClure W . Rate-limiting steps in RNA chain initiation. Proc Natl Acad Sci U S A. 1980; 77(10):5634-8. PMC: 350123. DOI: 10.1073/pnas.77.10.5634. View

12.

Lee N, Gielow W, Wallace R . Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. Proc Natl Acad Sci U S A. 1981; 78(2):752-6. PMC: 319880. DOI: 10.1073/pnas.78.2.752. View

13.

Kingston R, Gutell R, Taylor A, CHAMBERLIN M . Transcriptional mapping of plasmid pKK3535. Quantitation of the effect of guanosine tetraphosphate on binding to the rrnB promoters and a lambda promoter with sequence homologies in the CII binding region. J Mol Biol. 1981; 146(4):433-49. DOI: 10.1016/0022-2836(81)90041-3. View

14.

Russell D, Bennett G . Characterization of the beta-lactamase promoter of pBR322. Nucleic Acids Res. 1981; 9(11):2517-33. PMC: 326868. DOI: 10.1093/nar/9.11.2517. View

15.

Kanazawa H, Mabuchi K, Futai M . Nucleotide sequence of the promoter region of the gene cluster for proton-translocating ATPase from Escherichia coli and identification of the active promotor. Biochem Biophys Res Commun. 1982; 107(2):568-75. DOI: 10.1016/0006-291x(82)91529-7. View

16.

Travers A, Lamond A, Mace H . PpGpp regulates the binding of two RNA polymerase molecules to the tyrT promoter. Nucleic Acids Res. 1982; 10(16):5043-57. PMC: 320850. DOI: 10.1093/nar/10.16.5043. View

17.

Le Grice S, Sonenshein A . Interaction of Bacillus subtilis RNA polymerase with a chromosomal promoter. J Mol Biol. 1982; 162(3):551-64. DOI: 10.1016/0022-2836(82)90388-6. View

18.

Gourse R, Stark M, Dahlberg A . Regions of DNA involved in the stringent control of plasmid-encoded rRNA in vivo. Cell. 1983; 32(4):1347-54. DOI: 10.1016/0092-8674(83)90315-x. View

19.

Shaner S, Melancon P, Lee K, Burgess R, Record Jr M . Ion effects on the aggregation and DNA-binding reactions of Escherichia coli RNA polymerase. Cold Spring Harb Symp Quant Biol. 1983; 47 Pt 1:463-72. DOI: 10.1101/sqb.1983.047.01.055. View

20.

Gourse R, Nomura M . Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons. Cell. 1983; 33(3):865-76. DOI: 10.1016/0092-8674(83)90029-6. View