Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Jul 1;25(13):2640–2647. doi: 10.1093/nar/25.13.2640

Physical interference between escherichia coli RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation.

T Kubori 1, N Shimamoto 1
PMCID: PMC146789  PMID: 9185576

Abstract

Transcription initiation is accompanied with iterative synthesis and release of short transcripts. The molar ratio of enzyme to template was found to be critical for the amounts and distribution of the abortive products synthesized by Escherichia coli RNA polymerase from several promoters. At a high ratio abortive synthesis of 4-8 nt were enhanced at thelambda P R promoter. Removing excess RNA polymerase just before initiation, achieved by washing immobilized transcription complexes, prevented this enhancement. At this high ratio synthesis of an unexpected 6 nt transcript was enhanced when the enzyme stalled at position +32, but not when it stalled at position +73. This transcript had misincorporations at its fifth and sixth positions, probably due to slippage. Hydroxyl radical footprinting of the complex stalled at +32 in the presence of excess enzyme showed that more than one molecule of RNA polymerase was tandemly bound to the same DNA. These results suggest that: (i) when RNA polymerase molecules are tandemly transcribing the same DNA, transient collisions enhance abortive synthesis by the trailing molecule; (ii) when the leading polymerase stalled in the initially transcribed region blocks progression of the trailing polymerase, the latter can commit misincorporations, probably due to slippage synthesis.

Full Text

The Full Text of this article is available as a PDF (209.7 KB).

Selected References

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

  1. Ackerman S., Bunick D., Zandomeni R., Weinmann R. RNA polymerase II ternary transcription complexes generated in vitro. Nucleic Acids Res. 1983 Sep 10;11(17):6041–6064. doi: 10.1093/nar/11.17.6041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Borukhov S., Polyakov A., Nikiforov V., Goldfarb A. GreA protein: a transcription elongation factor from Escherichia coli. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8899–8902. doi: 10.1073/pnas.89.19.8899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borukhov S., Sagitov V., Goldfarb A. Transcript cleavage factors from E. coli. Cell. 1993 Feb 12;72(3):459–466. doi: 10.1016/0092-8674(93)90121-6. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Condon C., French S., Squires C., Squires C. L. Depletion of functional ribosomal RNA operons in Escherichia coli causes increased expression of the remaining intact copies. EMBO J. 1993 Nov;12(11):4305–4315. doi: 10.1002/j.1460-2075.1993.tb06115.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Craig M. L., Suh W. C., Record M. T., Jr HO. and DNase I probing of E sigma 70 RNA polymerase--lambda PR promoter open complexes: Mg2+ binding and its structural consequences at the transcription start site. Biochemistry. 1995 Dec 5;34(48):15624–15632. doi: 10.1021/bi00048a004. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Erie D. A., Hajiseyedjavadi O., Young M. C., von Hippel P. H. Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription. Science. 1993 Nov 5;262(5135):867–873. doi: 10.1126/science.8235608. [DOI] [PubMed] [Google Scholar]
  10. Feng G. H., Lee D. N., Wang D., Chan C. L., Landick R. GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure. J Biol Chem. 1994 Sep 2;269(35):22282–22294. [PubMed] [Google Scholar]
  11. Fujioka M., Hirata T., Shimamoto N. Requirement for the beta,gamma-pyrophosphate bond of ATP in a stage between transcription initiation and elongation by Escherichia coli RNA polymerase. Biochemistry. 1991 Feb 19;30(7):1801–1807. doi: 10.1021/bi00221a011. [DOI] [PubMed] [Google Scholar]
  12. Furuichi Y. Allosteric stimulatory effect of S-adenosylmethionine on the RNA polymerase in cytoplasmic polyhedrosis virus. A model for the positive control of eukaryotic transcription. J Biol Chem. 1981 Jan 10;256(1):483–493. [PubMed] [Google Scholar]
  13. Gonzalez N., Wiggs J., Chamberlin M. J. A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase. Arch Biochem Biophys. 1977 Aug;182(2):404–408. doi: 10.1016/0003-9861(77)90521-5. [DOI] [PubMed] [Google Scholar]
  14. Grayhack E. J., Yang X. J., Lau L. F., Roberts J. W. Phage lambda gene Q antiterminator recognizes RNA polymerase near the promoter and accelerates it through a pause site. Cell. 1985 Aug;42(1):259–269. doi: 10.1016/s0092-8674(85)80121-5. [DOI] [PubMed] [Google Scholar]
  15. Guo H. C., Roberts J. W. Heterogeneous initiation due to slippage at the bacteriophage 82 late gene promoter in vitro. Biochemistry. 1990 Nov 27;29(47):10702–10709. doi: 10.1021/bi00499a019. [DOI] [PubMed] [Google Scholar]
  16. Harley C. B., Lawrie J., Boyer H. W., Hedgpeth J. Reiterative copying by E.coli RNA polymerase during transcription initiation of mutant pBR322 tet promoters. Nucleic Acids Res. 1990 Feb 11;18(3):547–552. doi: 10.1093/nar/18.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Igarashi K., Ishihama A. Bipartite functional map of the E. coli RNA polymerase alpha subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell. 1991 Jun 14;65(6):1015–1022. doi: 10.1016/0092-8674(91)90553-b. [DOI] [PubMed] [Google Scholar]
  18. Ishihama A. Protein-protein communication within the transcription apparatus. J Bacteriol. 1993 May;175(9):2483–2489. doi: 10.1128/jb.175.9.2483-2489.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jacques J. P., Susskind M. M. Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter. Genes Dev. 1990 Oct;4(10):1801–1810. doi: 10.1101/gad.4.10.1801. [DOI] [PubMed] [Google Scholar]
  20. Kabata H., Kurosawa O., Arai I., Washizu M., Margarson S. A., Glass R. E., Shimamoto N. Visualization of single molecules of RNA polymerase sliding along DNA. Science. 1993 Dec 3;262(5139):1561–1563. doi: 10.1126/science.8248804. [DOI] [PubMed] [Google Scholar]
  21. Kubori T., Shimamoto N. A branched pathway in the early stage of transcription by Escherichia coli RNA polymerase. J Mol Biol. 1996 Mar 1;256(3):449–457. doi: 10.1006/jmbi.1996.0100. [DOI] [PubMed] [Google Scholar]
  22. Kubori T., Shimamoto N. Kinetics of transcription in a minute column. Nucleic Acids Res. 1996 Apr 1;24(7):1380–1381. doi: 10.1093/nar/24.7.1380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Levin J. R., Krummel B., Chamberlin M. J. Isolation and properties of transcribing ternary complexes of Escherichia coli RNA polymerase positioned at a single template base. J Mol Biol. 1987 Jul 5;196(1):85–100. doi: 10.1016/0022-2836(87)90512-2. [DOI] [PubMed] [Google Scholar]
  24. Machida C., Machida Y., Ohtsubo E. Both inverted repeat sequences located at the ends of IS1 provide promoter functions. J Mol Biol. 1984 Aug 5;177(2):247–267. doi: 10.1016/0022-2836(84)90455-8. [DOI] [PubMed] [Google Scholar]
  25. McAllister W. T., Küpper H., Bautz E. K. Kinetics of transcription by the bacteriophage-T3 RNA polymerase in vitro. Eur J Biochem. 1973 May 2;34(3):489–501. doi: 10.1111/j.1432-1033.1973.tb02785.x. [DOI] [PubMed] [Google Scholar]
  26. McClure W. R., Cech C. L. On the mechanism of rifampicin inhibition of RNA synthesis. J Biol Chem. 1978 Dec 25;253(24):8949–8956. [PubMed] [Google Scholar]
  27. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  28. Metzger W., Schickor P., Heumann H. A cinematographic view of Escherichia coli RNA polymerase translocation. EMBO J. 1989 Sep;8(9):2745–2754. doi: 10.1002/j.1460-2075.1989.tb08416.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Metzger W., Schickor P., Meier T., Werel W., Heumann H. Nucleation of RNA chain formation by Escherichia coli DNA-dependent RNA polymerase. J Mol Biol. 1993 Jul 5;232(1):35–49. doi: 10.1006/jmbi.1993.1368. [DOI] [PubMed] [Google Scholar]
  30. Mita B. C., Tang Y., deHaseth P. L. Interference of PR-bound RNA polymerase with open complex formation at PRM is relieved by a 10-base pair deletion between the two promoters. J Biol Chem. 1995 Dec 22;270(51):30428–30433. doi: 10.1074/jbc.270.51.30428. [DOI] [PubMed] [Google Scholar]
  31. Nam S. C., Kang C. W. Transcription initiation site selection and abortive initiation cycling of phage SP6 RNA polymerase. J Biol Chem. 1988 Dec 5;263(34):18123–18127. [PubMed] [Google Scholar]
  32. Ponnambalam S., Busby S. RNA polymerase molecules initiating transcription at tandem promoters can collide and cause premature transcription termination. FEBS Lett. 1987 Feb 9;212(1):21–27. doi: 10.1016/0014-5793(87)81549-1. [DOI] [PubMed] [Google Scholar]
  33. Qi F., Turnbough C. L., Jr Regulation of codBA operon expression in Escherichia coli by UTP-dependent reiterative transcription and UTP-sensitive transcriptional start site switching. J Mol Biol. 1995 Dec 8;254(4):552–565. doi: 10.1006/jmbi.1995.0638. [DOI] [PubMed] [Google Scholar]
  34. Shaner S. L., Piatt D. M., Wensley C. G., Yu H., Burgess R. R., Record M. T., Jr Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme. Biochemistry. 1982 Oct 26;21(22):5539–5551. doi: 10.1021/bi00265a025. [DOI] [PubMed] [Google Scholar]
  35. Shimamoto N., Wu F. Y., Wu C. W. Mechanism of ribonucleic acid chain initiation. Molecular pulse-labeling study of ribonucleic acid syntheses on T7 deoxyribonucleic acid template. Biochemistry. 1981 Aug 4;20(16):4745–4755. doi: 10.1021/bi00519a034. [DOI] [PubMed] [Google Scholar]
  36. Straney D. C., Crothers D. M. A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter. J Mol Biol. 1987 Jan 20;193(2):267–278. doi: 10.1016/0022-2836(87)90218-x. [DOI] [PubMed] [Google Scholar]
  37. Tullius T. D., Dombroski B. A. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5469–5473. doi: 10.1073/pnas.83.15.5469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Xiong X. F., Reznikoff W. S. Transcriptional slippage during the transcription initiation process at a mutant lac promoter in vivo. J Mol Biol. 1993 Jun 5;231(3):569–580. doi: 10.1006/jmbi.1993.1310. [DOI] [PubMed] [Google Scholar]
  39. Yamakawa M., Furuichi Y., Nakashima K., LaFiandra A. J., Shatkin A. J. Excess synthesis of viral mRNA 5-terminal oligonucleotides by reovirus transcriptase. J Biol Chem. 1981 Jun 25;256(12):6507–6514. [PubMed] [Google Scholar]
  40. von Hippel P. H., Bear D. G., Morgan W. D., McSwiggen J. A. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem. 1984;53:389–446. doi: 10.1146/annurev.bi.53.070184.002133. [DOI] [PubMed] [Google Scholar]

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

RESOURCES