Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Jul 25;20(14):3591–3598. doi: 10.1093/nar/20.14.3591

Transcription by an immobilized RNA polymerase from bacteriophage T7 and the topology of transcription.

P R Cook 1, F Gove 1
PMCID: PMC334006  PMID: 1641325

Abstract

It is often assumed that a polymerase moves along the template as it synthesizes RNA. However, a polymerase that tracks along a helical strand will generate a transcript that is entwined about the template. No such interlocking results if the polymerase is immobile and the template moves past it. Therefore we investigated whether immobilization inhibits the RNA polymerase of T7 bacteriophage using a hybrid protein, in which the polymerase is connected through a peptide linker to an immobilizing domain, which in turn was attached through an antibody to protein A covalently linked to plastic beads. Polymerase could be released by cleaving the linker with a protease, factor Xa. Comparison of the activity of the bound and free enzymes showed that immobilization reduced the rate of initiation about fivefold. However, when re-initiation was eliminated by removing excess template, immobilization was found to have little effect on the rate of elongation. Perhaps the untwining problem is sidestepped in vivo by immobilizing the polymerase.

Full text

PDF
3592

Images in this article

3594Image
on p.3594
3595Image
on p.3595
3596Image
on p.3596

Selected References

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

  1. Arndt K. M., Chamberlin M. J. RNA chain elongation by Escherichia coli RNA polymerase. Factors affecting the stability of elongating ternary complexes. J Mol Biol. 1990 May 5;213(1):79–108. doi: 10.1016/S0022-2836(05)80123-8. [DOI] [PubMed] [Google Scholar]
  2. Chamberlin M., Kingston R., Gilman M., Wiggs J., deVera A. Isolation of bacterial and bacteriophage RNA polymerases and their use in synthesis of RNA in vitro. Methods Enzymol. 1983;101:540–568. doi: 10.1016/0076-6879(83)01037-x. [DOI] [PubMed] [Google Scholar]
  3. Cook P. R. How mobile are active RNA polymerases? J Cell Sci. 1990 Jun;96(Pt 2):189–192. doi: 10.1242/jcs.96.2.189. [DOI] [PubMed] [Google Scholar]
  4. Cook P. R. The nucleoskeleton and the topology of transcription. Eur J Biochem. 1989 Nov 20;185(3):487–501. doi: 10.1111/j.1432-1033.1989.tb15141.x. [DOI] [PubMed] [Google Scholar]
  5. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dröge P., Nordheim A. Transcription-induced conformational change in a topologically closed DNA domain. Nucleic Acids Res. 1991 Jun 11;19(11):2941–2946. doi: 10.1093/nar/19.11.2941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  8. Duplay P., Bedouelle H., Fowler A., Zabin I., Saurin W., Hofnung M. Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12. J Biol Chem. 1984 Aug 25;259(16):10606–10613. [PubMed] [Google Scholar]
  9. Duplay P., Bedouelle H., Fowler A., Zabin I., Saurin W., Hofnung M. Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12. J Biol Chem. 1984 Aug 25;259(16):10606–10613. [PubMed] [Google Scholar]
  10. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ikeda R. A., Richardson C. C. Enzymatic properties of a proteolytically nicked RNA polymerase of bacteriophage T7. J Biol Chem. 1987 Mar 15;262(8):3790–3799. [PubMed] [Google Scholar]
  12. Jackson D. A., McCready S. J., Cook P. R. RNA is synthesized at the nuclear cage. Nature. 1981 Aug 6;292(5823):552–555. doi: 10.1038/292552a0. [DOI] [PubMed] [Google Scholar]
  13. Liu L. F., Wang J. C. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. doi: 10.1073/pnas.84.20.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McClelland M., Hanish J., Nelson M., Patel Y. KGB: a single buffer for all restriction endonucleases. Nucleic Acids Res. 1988 Jan 11;16(1):364–364. doi: 10.1093/nar/16.1.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ostrander E. A., Benedetti P., Wang J. C. Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase. Science. 1990 Sep 14;249(4974):1261–1265. doi: 10.1126/science.2399463. [DOI] [PubMed] [Google Scholar]
  16. Pettijohn D. E. Histone-like proteins and bacterial chromosome structure. J Biol Chem. 1988 Sep 15;263(26):12793–12796. [PubMed] [Google Scholar]
  17. Pruss G. J., Drlica K. DNA supercoiling and prokaryotic transcription. Cell. 1989 Feb 24;56(4):521–523. doi: 10.1016/0092-8674(89)90574-6. [DOI] [PubMed] [Google Scholar]
  18. Schafer D. A., Gelles J., Sheetz M. P., Landick R. Transcription by single molecules of RNA polymerase observed by light microscopy. Nature. 1991 Aug 1;352(6334):444–448. doi: 10.1038/352444a0. [DOI] [PubMed] [Google Scholar]
  19. Tsao Y. P., Wu H. Y., Liu L. F. Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies. Cell. 1989 Jan 13;56(1):111–118. doi: 10.1016/0092-8674(89)90989-6. [DOI] [PubMed] [Google Scholar]
  20. Wu H. Y., Shyy S. H., Wang J. C., Liu L. F. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. doi: 10.1016/0092-8674(88)90163-8. [DOI] [PubMed] [Google Scholar]
  21. Zawadzki V., Gross H. J. Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res. 1991 Apr 25;19(8):1948–1948. doi: 10.1093/nar/19.8.1948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. ten Heggeler-Bordier B., Wahli W., Adrian M., Stasiak A., Dubochet J. The apical localization of transcribing RNA polymerases on supercoiled DNA prevents their rotation around the template. EMBO J. 1992 Feb;11(2):667–672. doi: 10.1002/j.1460-2075.1992.tb05098.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES