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 phi 29 activates the vir al late A3 promoter mainly by stabilizing the binding of RNA polymerase (RN AP) 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 subuni t. Several acidic residues of the alpha -CTD, considered as plausible targe ts for p4 residue Arg120, were individually changed into alanine. In additi on, a truncated alpha subunit lacking the last four residues, two of which are acidic, was obtained. The modified alpha subunits were purified and rec onstituted 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 s everal other promoters. Interestingly, protein p4 could stabilize the modif ied RNAP at the A3 promoter as a closed complex, although the open complexe s formed were unstable and did not proceed to elongation complexes. Our res ults indicate that the change of the alpha residue Glu297 into Ala destabil izes the open complexes formed at this promoter, but not at other promoters . Considered in the context of earlier findings indicating that the RNAP al pha -CTD may participate in the transition from closed to intermediate comp lexes at some other promoters, the new results expand and clarify our view of its role in transcription initiation. (C) 2001 Academic Press.