A Mutation in the C-terminal domain of the RNA polymerase alpha subunit that destabilizes the open complexes formed at the phage phi 29 late A3 promoter.
Regulatory protein p4 from Bacillus subtilis phage phi29 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 alpha subunit. Several acidic residues of the alpha-CTD, considered as plausible targets for p4 residue Arg120, were individually changed into alanine. In addition, a truncated alpha subunit lacking the last four residues, two of which are acidic, was obtained. The modified alpha subunits were purified 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 alpha subunit was changed to Ala, a modification that did not impair transcription from several other promoters. Interestingly, protein p4 could stabilize the modified 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 alpha residue Glu297 into Ala destabilizes the open complexes formed at this promoter, but not at other promoters. Considered in the context of earlier findings indicating that the RNAP alpha-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.
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
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Monsalve
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D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics* / Q000821:virology; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000378:metabolism
D014157:Transcription Factors; D014764:Viral Proteins; C053987:p4 protein, Bacteriophage phi 29; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit