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 (original) (raw)
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Journal of Molecular Biology, 1998
Regulatory protein p4 from Bacillus subtilis phage È29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase a subunit. This interaction stabilizes the RNA polymerase at the promoter in such a way that promoter clearance is prevented. Here, the binding of protein p4 to the A2c promoter has been studied. In the absence of RNA polymerase, protein p4 was found to bind with low af®nity to a site centered at position À39 relative to the transcription start site. When RNA polymerase was present, protein p4 was displaced from this site and bound instead to a different target centered at position À71. Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase a-subunit. Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high af®nity. A mutational analysis revealed that the site at À71 is critical for a stable interaction between protein p4 and RNA polymerase, and for ef®cient repression, whereas mutation of the site at À39 had only a small effect on repression ef®ciency. Therefore, RNA polymerase plays an active role in the repression mechanism by stabilizing the repressor at the promoter, generating a nucleoprotein complex that is too stable to allow promoter clearance.
Molecular Microbiology, 1996
Phage φ29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing σA-RNA polymerase at the promoter as a closed complex. Activation requires interaction between both proteins. Protein p4 bends the DNA upon binding. We have performed a detailed mutagenesis study of the carboxyl end of the protein, which is involved in both transcription activation and DNA bending. The results indicate that Arg-120 is the most critical residue for activation, probably mediating the interaction with RNA polymerase. Several basic residues have been identified, including Arg-120, that contribute to maintenance of the DNA bending, probably via electrostatic interactions with the DNA backbone. The degree or stability of the induced bend apparently relies on the additive contribution of all basic residues of the carboxyl end of the protein. Therefore, the activation and DNA bending surfaces overlap, and Arg-120 should interact with both DNA and RNA polymerase. As we show that protein p4 is a dimer in solution, and is bound to DNA as a tetramer, the results suggest a model in which two of the p4 subunits interact with the DNA, bending it, while the other two subunits remain accessible to interact with RNA polymerase.
Journal of Molecular Biology, 2001
Regulatory protein p4 from Bacillus subtilis phage f29 activates the viral late A3 promoter mainly by stabilizing the binding of RNA polymerase (RNAP) to it as a closed complex. This requires an interaction between protein p4 residue Arg120 and the C-terminal domain (CTD) of the RNAP a subunit. Several acidic residues of the a-CTD, considered as plausible targets for p4 residue Arg120, were individually changed into alanine. In addition, a truncated a subunit lacking the last four residues, two of which are acidic, was obtained. The modi®ed a subunits were puri®ed and reconstituted into RNAP holoenzyme in vitro. Protein p4 was found to be unable to activate the late A3 promoter when residue Glu297 of the a subunit was changed to Ala, a modi®cation that did not impair transcription from several other promoters. Interestingly, protein p4 could stabilize the modi®ed RNAP at the A3 promoter as a closed complex, although the open complexes formed were unstable and did not proceed to elongation complexes. Our results indicate that the change of the a residue Glu297 into Ala destabilizes the open complexes formed at this promoter, but not at other promoters. Considered in the context of earlier ®ndings indicating that the RNAP a-CTD may participate in the transition from closed to intermediate complexes at some other promoters, the new results expand and clarify our view of its role in transcription initiation.
Distinct pathways of RNA polymerase regulation by a phage-encoded factor
Proceedings of the National Academy of Sciences of the United States of America, 2015
Transcription antitermination is a common strategy of gene expression regulation, but only a few transcription antitermination factors have been studied in detail. Here, we dissect the transcription antitermination mechanism of Xanthomonas oryzae virus Xp10 protein p7, which binds host RNA polymerase (RNAP) and regulates both transcription initiation and termination. We show that p7 suppresses intrinsic termination by decreasing RNAP pausing and increasing the transcription complex stability, in cooperation with host-encoded factor NusA. Uniquely, the antitermination activity of p7 depends on the ω subunit of the RNAP core and is modulated by ppGpp. In contrast, the inhibition of transcription initiation by p7 does not require ω but depends on other RNAP sites. Our results suggest that p7, a bifunctional transcription factor, uses distinct mechanisms to control different steps of transcription. We propose that regulatory functions of the ω subunit revealed by our analysis may extend...
Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein
Journal of Molecular Biology, 2011
E. coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). Gp2 inhibits formation of open promoter complex by binding to the β′ jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β′ jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the −10 promoter element. The inhibition of RNAP interactions with singlestranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β′ jaw.
Journal of Molecular Biology, 1999
The transcription program of the Bacillus phage GA-1, a distant relative of phage È29, has been studied. Transcription of the GA-1 genome occurred in two stages, early and late. Early genes were expressed from two promoters equivalent to the È29 A2b and A2c promoters, whereas late transcription started at a site equivalent to the È29 late A3 promoter. The activity of the GA-1 early A2b and A2c promoters diminished 10 minutes after infection, a time at which expression of the late promoter increased signi®cantly. The switch from early to late transcription required protein synthesis, suggesting the need for viral protein(s). An open reading frame was found in the GA-1 genome coding for a protein showing a 53 % similarity to È29 regulatory protein p4, and was named p4 G . In È29, protein p4 represses the early A2b and A2c promoters and activates the late A3 promoter by recruiting RNA polymerase to it. A binding site for protein p4 G was localized upstream from the GA-1 late A3 promoter, overlapping with the early A2b promoter. In vitro, protein p4 G prevented the binding of RNA polymerase to the GA-1 early A2b promoter but, unlike in È29, had no effect on the expression of the late A3 promoter: RNA polymerase could ef®ciently bind and initiate transcription from the A3 promoter in the absence of protein p4 G . Therefore, activation of late transcription occurs differently in GA-1 and È29. We propose that protein p4 G is an anti-repressor which inhibits the binding to the late promoter of an unknown repressor factor present in the host strain.