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. 1996 Sep 3;93(18):9408–9413. doi: 10.1073/pnas.93.18.9408

Distortion in the spacer region of Pm during activation of middle transcription of phage Mu.

I Artsimovitch 1, M Kahmeyer-Gabbe 1, M M Howe 1
PMCID: PMC38441  PMID: 8790343

Abstract

Transcription from the middle promoter, Pm, of phage Mu is initiated by Escherichia coli RNA polymerase holoenzyme (E sigma 70; RNAP) and the phage-encoded activator, Mor. Point mutations in the spacer region between the -10 hexamer and the Mor binding site result in changes of promoter activity in vivo. These mutations are located at the junction between a rigid T-tract and adjacent, potentially deformable G + C-rich DNA segment, suggesting that deformation of the spacer region may play a role in the transcriptional activation of Pm. This prediction was tested by using dimethyl sulfate and potassium permanganate footprinting analyses. Helical distortion involving strand separation was detected at positions -32 to -34, close to the predicted interface between Mor and RNAP. Promoter mutants in which this distortion was not detected exhibited a lack of melting in the -12 to -1 region and reduced promoter activity in vivo. We propose that complexes containing the distortion represent stressed intermediates rather than stable open complexes and thus can be envisaged as a transition state in the kinetic pathway of Pm activation in which stored torsional energy could be used to facilitate melting around the transcription start point.

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Selected References

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

  1. Artsimovitch I., Howe M. M. Transcription activation by the bacteriophage Mu Mor protein: analysis of promoter mutations in Pm identifies a new region required for promoter function. Nucleic Acids Res. 1996 Feb 1;24(3):450–457. doi: 10.1093/nar/24.3.450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auble D. T., Allen T. L., deHaseth P. L. Promoter recognition by Escherichia coli RNA polymerase. Effects of substitutions in the spacer DNA separating the -10 and -35 regions. J Biol Chem. 1986 Aug 25;261(24):11202–11206. [PubMed] [Google Scholar]
  3. Auble D. T., deHaseth P. L. Promoter recognition by Escherichia coli RNA polymerase. Influence of DNA structure in the spacer separating the -10 and -35 regions. J Mol Biol. 1988 Aug 5;202(3):471–482. doi: 10.1016/0022-2836(88)90279-3. [DOI] [PubMed] [Google Scholar]
  4. Ayers D. G., Auble D. T., deHaseth P. L. Promoter recognition by Escherichia coli RNA polymerase. Role of the spacer DNA in functional complex formation. J Mol Biol. 1989 Jun 20;207(4):749–756. doi: 10.1016/0022-2836(89)90241-6. [DOI] [PubMed] [Google Scholar]
  5. Borowiec J. A., Gralla J. D. High-resolution analysis of lac transcription complexes inside cells. Biochemistry. 1986 Sep 9;25(18):5051–5057. doi: 10.1021/bi00366a012. [DOI] [PubMed] [Google Scholar]
  6. Buc H., McClure W. R. Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps. Biochemistry. 1985 May 21;24(11):2712–2723. doi: 10.1021/bi00332a018. [DOI] [PubMed] [Google Scholar]
  7. Busby S., Ebright R. H. Promoter structure, promoter recognition, and transcription activation in prokaryotes. Cell. 1994 Dec 2;79(5):743–746. doi: 10.1016/0092-8674(94)90063-9. [DOI] [PubMed] [Google Scholar]
  8. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cowing D. W., Mecsas J., Record M. T., Jr, Gross C. A. Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor sigma 32 at the groE promoter. J Mol Biol. 1989 Dec 5;210(3):521–530. doi: 10.1016/0022-2836(89)90128-9. [DOI] [PubMed] [Google Scholar]
  11. Crothers D. M., Haran T. E., Nadeau J. G. Intrinsically bent DNA. J Biol Chem. 1990 May 5;265(13):7093–7096. [PubMed] [Google Scholar]
  12. Fong R. S., Woody S., Gussin G. N. Modulation of P(RM) activity by the lambda PR promoter in both the presence and absence of repressor. J Mol Biol. 1993 Aug 5;232(3):792–804. doi: 10.1006/jmbi.1993.1432. [DOI] [PubMed] [Google Scholar]
  13. Howe M. M. Prophage deletion mapping of bacteriophage Mu-1. Virology. 1973 Jul;54(1):93–101. doi: 10.1016/0042-6822(73)90118-9. [DOI] [PubMed] [Google Scholar]
  14. Jiang Y., Triezenberg S. J., Gralla J. D. Defective transcriptional activation by diverse VP16 mutants associated with a common inability to form open promoter complexes. J Biol Chem. 1994 Feb 25;269(8):5505–5508. [PubMed] [Google Scholar]
  15. Kahmeyer-Gabbe M., Howe M. M. Regulatory factors acting at the bacteriophage Mu middle promoter. J Bacteriol. 1996 Mar;178(6):1585–1592. doi: 10.1128/jb.178.6.1585-1592.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kolb A., Busby S., Buc H., Garges S., Adhya S. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993;62:749–795. doi: 10.1146/annurev.bi.62.070193.003533. [DOI] [PubMed] [Google Scholar]
  17. Kumar A., Grimes B., Fujita N., Makino K., Malloch R. A., Hayward R. S., Ishihama A. Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation. J Mol Biol. 1994 Jan 14;235(2):405–413. doi: 10.1006/jmbi.1994.1001. [DOI] [PubMed] [Google Scholar]
  18. Marrs C. F., Howe M. M. Kinetics and regulation of transcription of bacteriophage Mu. Virology. 1990 Jan;174(1):192–203. doi: 10.1016/0042-6822(90)90068-3. [DOI] [PubMed] [Google Scholar]
  19. Mathee K., Howe M. M. Bacteriophage Mu Mor protein requires sigma 70 to activate the Mu middle promoter. J Bacteriol. 1993 Sep;175(17):5314–5323. doi: 10.1128/jb.175.17.5314-5323.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mathee K., Howe M. M. Identification of a positive regulator of the Mu middle operon. J Bacteriol. 1990 Dec;172(12):6641–6650. doi: 10.1128/jb.172.12.6641-6650.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maxam A. M., 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. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Mustaev A., Zaychikov E., Severinov K., Kashlev M., Polyakov A., Nikiforov V., Goldfarb A. Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12036–12040. doi: 10.1073/pnas.91.25.12036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Owen R. J., Borman P. A rapid biochemical method for purifying high molecular weight bacterial chromosomal DNA for restriction enzyme analysis. Nucleic Acids Res. 1987 Apr 24;15(8):3631–3631. doi: 10.1093/nar/15.8.3631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ramstein J., Lavery R. Energetic coupling between DNA bending and base pair opening. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7231–7235. doi: 10.1073/pnas.85.19.7231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roe J. H., Burgess R. R., Record M. T., Jr Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening. J Mol Biol. 1985 Aug 5;184(3):441–453. doi: 10.1016/0022-2836(85)90293-1. [DOI] [PubMed] [Google Scholar]
  27. Ryu S., Garges S., Adhya S. An arcane role of DNA in transcription activation. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8582–8586. doi: 10.1073/pnas.91.18.8582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sasse-Dwight S., Gralla J. D. Footprinting protein-DNA complexes in vivo. Methods Enzymol. 1991;208:146–168. doi: 10.1016/0076-6879(91)08012-7. [DOI] [PubMed] [Google Scholar]
  29. Siebenlist U. RNA polymerase unwinds an 11-base pair segment of a phage T7 promoter. Nature. 1979 Jun 14;279(5714):651–652. doi: 10.1038/279651a0. [DOI] [PubMed] [Google Scholar]
  30. Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
  31. Sollner-Webb B., Reeder R. H. The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis. Cell. 1979 Oct;18(2):485–499. doi: 10.1016/0092-8674(79)90066-7. [DOI] [PubMed] [Google Scholar]
  32. Spassky A., Kirkegaard K., Buc H. Changes in the DNA structure of the lac UV5 promoter during formation of an open complex with Escherichia coli RNA polymerase. Biochemistry. 1985 May 21;24(11):2723–2731. doi: 10.1021/bi00332a019. [DOI] [PubMed] [Google Scholar]
  33. Stefano J. E., Gralla J. D. Spacer mutations in the lac ps promoter. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1069–1072. doi: 10.1073/pnas.79.4.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stoddard S. F., Howe M. M. Characterization of the C operon transcript of bacteriophage Mu. J Bacteriol. 1990 Jan;172(1):361–371. doi: 10.1128/jb.172.1.361-371.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Straney D. C., Crothers D. M. Intermediates in transcription initiation from the E. coli lac UV5 promoter. Cell. 1985 Dec;43(2 Pt 1):449–459. doi: 10.1016/0092-8674(85)90175-8. [DOI] [PubMed] [Google Scholar]
  36. Toussaint A., Lecocq J. P. Sensitivity of bacteriophage Mu-1 development to rifampicin and streptolydigin. Mol Gen Genet. 1974 Mar 14;129(2):185–188. doi: 10.1007/BF00268631. [DOI] [PubMed] [Google Scholar]
  37. Travers A. A. Structure and function of E. coli promoter DNA. CRC Crit Rev Biochem. 1987;22(3):181–219. doi: 10.3109/10409238709101483. [DOI] [PubMed] [Google Scholar]
  38. Wijffelman C., Gassler M., Stevens W. F., van de Putte P. On the control of transcription of bacteriophage Mu. Mol Gen Genet. 1974;131(2):85–96. doi: 10.1007/BF00266145. [DOI] [PubMed] [Google Scholar]
  39. deHaseth P. L., Helmann J. D. Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase-induced strand separation of double helical DNA. Mol Microbiol. 1995 Jun;16(5):817–824. doi: 10.1111/j.1365-2958.1995.tb02309.x. [DOI] [PubMed] [Google Scholar]

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