Skip to main content
NCBI 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
. 1977 Sep;74(9):3740–3744. doi: 10.1073/pnas.74.9.3740

Electron microscope studies of transient complexes formed between Escherichia coli RNA polymerase holoenzyme and T7 DNA.

R C Williams, M J Chamberlin
PMCID: PMC431710  PMID: 333445

Abstract

Electron microscopy was used to study the formation of random complexes between Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a promoterless fragment (Mbo I-C) of bacteriophage T7 DNA, and to determine the location of the polymerase molecules bound at 3 degrees to the promoter-containing (Hinf)1100 fragment of the same DNA. The value of the Ka of random binding is about 3 times 10(4)M-1 when the enzyme is slowly diluted from its storage condition and is incubated with DNA for up to 2 min at 37 degrees. If dilution is rapid and occurs in a single step, or if incubation extends beyond 5 min, a substantial portion of RNA polymerase is converted to a form that binds randomly with a much greater affinity (about 10(8)M-1). Hence true random binding by RNA polymerase holoenzyme is much weaker than previously thought. However, great caution is required in assessing the extent of random binding where damage to the enzyme may occur. When RNApolymerase holoenzyme is incubated at 0 degrees with promoter-containing fragment (Hinf)1100, complexes form at the same promoter sites utilized at 37 degrees, although the highly stable "open" promoter complex is not formed under these conditions. However, the extent of binding is reduced as compared to promoter complexes formed at 37 degrees. This gives direct evidence for formation of complexes with promoter sites that have properties of the hypothetical "closed"complexes formed between RNA polymerase and duplex DNA.

Full text

PDF
3740

Images in this article

3741Image
on p.3741

Selected References

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

  1. Bordier C., Dubochet J. Electron microscopic localization of the binding sites of Escherichia coli RNA polymerase in the early promoter region of T7 DNA. Eur J Biochem. 1974 May 15;44(2):617–624. doi: 10.1111/j.1432-1033.1974.tb03519.x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Crawford L. V., Crawford E. M., Richardson J. P., Slayter H. S. The binding of RNA polymerase to polyoma and papilloma DNA. J Mol Biol. 1965 Dec;14(2):593–597. doi: 10.1016/s0022-2836(65)80209-1. [DOI] [PubMed] [Google Scholar]
  4. Dubochet J., Kellenberger E. Selective adsorption of particles to the supporting film and its consequences on particle counts in electron microscopy. Microsc Acta. 1972 Jul;72(2):119–130. [PubMed] [Google Scholar]
  5. Dunn J. J., Studier F. W. T7 early RNAs are generated by site-specific cleavages. Proc Natl Acad Sci U S A. 1973 May;70(5):1559–1563. doi: 10.1073/pnas.70.5.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hinkle D. C., Chamberlin M. J. Studies of the binding of Escherichia coli RNA polymerase to DNA. II. The kinetics of the binding reaction. J Mol Biol. 1972 Sep 28;70(2):187–195. doi: 10.1016/0022-2836(72)90532-3. [DOI] [PubMed] [Google Scholar]
  7. Hirsh J., Schleif R. High resolution electron microscopic studies of genetic regulation. J Mol Biol. 1976 Dec;108(2):471–490. doi: 10.1016/s0022-2836(76)80131-3. [DOI] [PubMed] [Google Scholar]
  8. Hsieh T., Wang J. C. Physicochomecial studies on interactions between DNA and RNA polymerase. Isolation and mapping of a T7 DNA fragment containing the early promoters for Escherichia coli RNA polymerase. Biochemistry. 1976 Dec 28;15(26):5776–5783. doi: 10.1021/bi00671a014. [DOI] [PubMed] [Google Scholar]
  9. Koller T., Sogo J. M., Bujard H. An electron microscopic method for studying nucleic acid-protein complexes. Visualization of RNA polymerase bound to the DNA of bacteriophages T7 and T3. Biopolymers. 1974 May;13(5):995–1009. doi: 10.1002/bip.1974.360130514. [DOI] [PubMed] [Google Scholar]
  10. Lescure B., Oudet P., Chambon P., Yaniv M. Transcription of polyoma virus DNA in vitro. Localization of Escherichia coli RNA polymerase initiation sites. J Mol Biol. 1976 Nov;108(1):83–97. doi: 10.1016/s0022-2836(76)80096-4. [DOI] [PubMed] [Google Scholar]
  11. Mangel W. F., Chamberlin M. J. Studies of ribonucleic acid chain initiation by Escherichia coli ribonucleic acid polymerase bound to T7 deoxyribonucleic acid. 3. The effect of temperature on ribonucleic acid chain initiation and on the conformation of binary complexes. J Biol Chem. 1974 May 25;249(10):3007–3013. [PubMed] [Google Scholar]
  12. 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]
  13. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  14. Minkley E. G., Pribnow D. Transcription of the early region of bacteriophage T7: selective initiation with dinucleotides. J Mol Biol. 1973 Jun 25;77(2):255–277. doi: 10.1016/0022-2836(73)90335-5. [DOI] [PubMed] [Google Scholar]
  15. Okamoto T., Sugimoto K., Sugisaki H., Takanami M. Studies on bacteriophage fd DNA. II. Localization of RNA initiation sites on the cleavage map of the fd genome. J Mol Biol. 1975 Jun 15;95(1):33–44. doi: 10.1016/0022-2836(75)90333-2. [DOI] [PubMed] [Google Scholar]
  16. Richardson J. P. The binding of RNA polymerase to DNA. J Mol Biol. 1966 Oct 28;21(1):83–114. doi: 10.1016/0022-2836(66)90081-7. [DOI] [PubMed] [Google Scholar]
  17. Saucier J. M., Wang J. C. Angular alteration of the DNA helix by E. coli RNA polymerase. Nat New Biol. 1972 Oct 11;239(93):167–170. doi: 10.1038/newbio239167a0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  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. Sternberger N., Stevens A. Studies of complexes of RNA polymerase and lambda DNA. Biochem Biophys Res Commun. 1966 Sep 22;24(6):937–942. doi: 10.1016/0006-291x(66)90340-8. [DOI] [PubMed] [Google Scholar]
  21. Williams R. C. Use of polylysine for adsorption of nuclei acids and enzymes to electron microscope specimen films. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2311–2315. doi: 10.1073/pnas.74.6.2311. [DOI] [PMC free article] [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