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Regulatory protein p4 from Bacillus subtilis phage φ29 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 α subunit. Several acidic residues of the α-CTD, considered as plausible targets for p4 residue Arg120, were individually changed into alanine. In addition, a truncated α subunit lacking the last four residues, two of which are acidic, was obtained. The modified α 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 α 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 …