Binding of phage phi29 protein p4 to the early A2c promoter: recruitment of a repressor by the RNA polymerase.
Regulatory protein p4 from Bacillus subtilis phage Phi29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase alpha 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 affinity to a site centered at position -39 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 -71. Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase alpha-subunit. Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high affinity. A mutational analysis revealed that the site at -71 is critical for a stable interaction between protein p4 and RNA polymerase, and for efficient repression, whereas mutation of the site at -39 had only a small effect on repression efficiency. 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.
Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049-Madrid, Spain.
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D001412:Bacillus subtilis / Q000235:genetics / Q000821:virology; D001435:Bacteriophages / Q000378:metabolism; D001483:Base Sequence; D018983:DNA Footprinting; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C053987:p4 protein, Bacteriophage phi 29; D012321:DNA-Directed RNA Polymerases