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. 1996 Jan 15;15(2):383–391.

Activation and repression of transcription at two different phage phi29 promoters are mediated by interaction of the same residues of regulatory protein p4 with RNA polymerase.

M Monsalve 1, M Mencia 1, F Rojo 1, M Salas 1
PMCID: PMC449953  PMID: 8617213

Abstract

Phage phi29 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Arg120 plays an essential role. We show that protein p4 represses the A2c promoter by binding to DNA immediately upstream from RNAP in a way that does not hinder RNAP binding; rather, the two proteins bind cooperatively to DNA. In the presence of protein p4, RNAP can form an initiated complex at the A2c promoter that generates short abortive transcripts, but cannot leave the promoter. Mutation of protein p4 residue Arg120, which relieves the contact between the two proteins, leads to a loss of repression. Therefore, the contact between protein p4 and RNAP through the protein p4 domain containing Arg120 can activate or repress transcription, depending on the promoter. The relative position of protein p4 and RNAP, which is different at each promoter, together with the distinct characteristics of the two promoters, may determine whether protein p4 activates or represses transcription.

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Selected References

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

  1. Barthelemy I., Salas M. Characterization of a new prokaryotic transcriptional activator and its DNA recognition site. J Mol Biol. 1989 Jul 20;208(2):225–232. doi: 10.1016/0022-2836(89)90384-7. [DOI] [PubMed] [Google Scholar]
  2. Carpousis A. J., Gralla J. D. Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry. 1980 Jul 8;19(14):3245–3253. doi: 10.1021/bi00555a023. [DOI] [PubMed] [Google Scholar]
  3. Carpousis A. J., Gralla J. D. Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation. Footprinting, methylation, and rifampicin-sensitivity changes accompanying transcription initiation. J Mol Biol. 1985 May 25;183(2):165–177. doi: 10.1016/0022-2836(85)90210-4. [DOI] [PubMed] [Google Scholar]
  4. Choy H. E., Adhya S. Control of gal transcription through DNA looping: inhibition of the initial transcribing complex. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11264–11268. doi: 10.1073/pnas.89.23.11264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eismann E. R., Müller-Hill B. lac repressor forms stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators. J Mol Biol. 1990 Jun 20;213(4):763–775. doi: 10.1016/S0022-2836(05)80262-1. [DOI] [PubMed] [Google Scholar]
  6. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Giladi H., Gottesman M., Oppenheim A. B. Integration host factor stimulates the phage lambda pL promoter. J Mol Biol. 1990 May 5;213(1):109–121. doi: 10.1016/S0022-2836(05)80124-X. [DOI] [PubMed] [Google Scholar]
  8. Hawley D. K., McClure W. R. Mechanism of activation of transcription initiation from the lambda PRM promoter. J Mol Biol. 1982 May 25;157(3):493–525. doi: 10.1016/0022-2836(82)90473-9. [DOI] [PubMed] [Google Scholar]
  9. Herbert M., Kolb A., Buc H. Overlapping promoters and their control in Escherichia coli: the gal case. Proc Natl Acad Sci U S A. 1986 May;83(9):2807–2811. doi: 10.1073/pnas.83.9.2807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Krummel B., Chamberlin M. J. RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes. Biochemistry. 1989 Sep 19;28(19):7829–7842. doi: 10.1021/bi00445a045. [DOI] [PubMed] [Google Scholar]
  11. Kuhnke G., Theres C., Fritz H. J., Ehring R. RNA polymerase and gal repressor bind simultaneously and with DNA bending to the control region of the Escherichia coli galactose operon. EMBO J. 1989 Apr;8(4):1247–1255. doi: 10.1002/j.1460-2075.1989.tb03498.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lane D., Prentki P., Chandler M. Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev. 1992 Dec;56(4):509–528. doi: 10.1128/mr.56.4.509-528.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lee J., Goldfarb A. lac repressor acts by modifying the initial transcribing complex so that it cannot leave the promoter. Cell. 1991 Aug 23;66(4):793–798. doi: 10.1016/0092-8674(91)90122-f. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Mencía M., Salas M., Rojo F. Residues of the Bacillus subtilis phage phi 29 transcriptional activator required both to interact with RNA polymerase and to activate transcription. J Mol Biol. 1993 Oct 20;233(4):695–704. doi: 10.1006/jmbi.1993.1546. [DOI] [PubMed] [Google Scholar]
  17. Menendez M., Kolb A., Buc H. A new target for CRP action at the malT promoter. EMBO J. 1987 Dec 20;6(13):4227–4234. doi: 10.1002/j.1460-2075.1987.tb02771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Monsalve M., Mencía M., Rojo F., Salas M. Transcription regulation in Bacillus subtilis phage phi 29: expression of the viral promoters throughout the infection cycle. Virology. 1995 Feb 20;207(1):23–31. doi: 10.1006/viro.1995.1048. [DOI] [PubMed] [Google Scholar]
  19. Morett E., Buck M. In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex. J Mol Biol. 1989 Nov 5;210(1):65–77. doi: 10.1016/0022-2836(89)90291-x. [DOI] [PubMed] [Google Scholar]
  20. Nuez B., Rojo F., Salas M. Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11401–11405. doi: 10.1073/pnas.89.23.11401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Popham D. L., Szeto D., Keener J., Kustu S. Function of a bacterial activator protein that binds to transcriptional enhancers. Science. 1989 Feb 3;243(4891):629–635. doi: 10.1126/science.2563595. [DOI] [PubMed] [Google Scholar]
  22. Rojo F., Alonso J. C. The beta recombinase from the Streptococcal plasmid pSM 19035 represses its own transcription by holding the RNA polymerase at the promoter region. Nucleic Acids Res. 1994 May 25;22(10):1855–1860. doi: 10.1093/nar/22.10.1855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rojo F., Nuez B., Mencía M., Salas M. The main early and late promoters of Bacillus subtilis phage phi 29 form unstable open complexes with sigma A-RNA polymerase that are stabilized by DNA supercoiling. Nucleic Acids Res. 1993 Feb 25;21(4):935–940. doi: 10.1093/nar/21.4.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rojo F., Salas M. A DNA curvature can substitute phage phi 29 regulatory protein p4 when acting as a transcriptional repressor. EMBO J. 1991 Nov;10(11):3429–3438. doi: 10.1002/j.1460-2075.1991.tb04907.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rojo F., Zaballos A., Salas M. Bend induced by the phage phi 29 transcriptional activator in the viral late promoter is required for activation. J Mol Biol. 1990 Feb 20;211(4):713–725. doi: 10.1016/0022-2836(90)90072-t. [DOI] [PubMed] [Google Scholar]
  26. Schlax P. J., Capp M. W., Record M. T., Jr Inhibition of transcription initiation by lac repressor. J Mol Biol. 1995 Jan 27;245(4):331–350. doi: 10.1006/jmbi.1994.0028. [DOI] [PubMed] [Google Scholar]
  27. Serrano M., Barthelemy I., Salas M. Transcription activation at a distance by phage phi 29 protein p4. Effect of bent and non-bent intervening DNA sequences. J Mol Biol. 1991 Jun 5;219(3):403–414. doi: 10.1016/0022-2836(91)90182-6. [DOI] [PubMed] [Google Scholar]
  28. Sogo J. M., Inciarte M. R., Corral J., Viñuela E., Salas M. RNA polymerase binding sites and transcription map of the DNA of Bacillus subtilis phage phi29. J Mol Biol. 1979 Feb 5;127(4):411–436. doi: 10.1016/0022-2836(79)90230-4. [DOI] [PubMed] [Google Scholar]
  29. Spassky A., Busby S., Buc H. On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions. EMBO J. 1984 Jan;3(1):43–50. doi: 10.1002/j.1460-2075.1984.tb01759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Straney D. C., Straney S. B., Crothers D. M. Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex. J Mol Biol. 1989 Mar 5;206(1):41–57. doi: 10.1016/0022-2836(89)90522-6. [DOI] [PubMed] [Google Scholar]
  31. Straney S. B., Crothers D. M. Lac repressor is a transient gene-activating protein. Cell. 1987 Dec 4;51(5):699–707. doi: 10.1016/0092-8674(87)90093-6. [DOI] [PubMed] [Google Scholar]
  32. Tsung K., Brissette R. E., Inouye M. Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5940–5944. doi: 10.1073/pnas.87.15.5940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Williams D. R., Motallebi-Veshareh M., Thomas C. M. Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase-promoter complexes. Nucleic Acids Res. 1993 Mar 11;21(5):1141–1148. doi: 10.1093/nar/21.5.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]

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