Protein p4 represses phage phi 29 A2c promoter by interacting with the alpha subunit of Bacillus subtilis RNA polymerase.
Regulatory protein p4 from Bacillus subtilis phage phi 29 represses the strong viral A2c promoter (PA2c) by preventing promoter clearance; it allows RNA polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached. Protein p4 binds at PA2c immediately upstream from RNA polymerase; repression involves a contact between both proteins that holds the RNA polymerase at the promoter. This contact is held mainly through p4 residue Arg120, which is also required for activation of the phi 29 late A3 promoter. We have investigated which region of RNA polymerase contacts protein p4 at PA2c. Promoter repression was impaired when a reconstituted RNA polymerase lacking the 15 C-terminal residues of the alpha subunit C-terminal domain was used; this polymerase was otherwise competent for transcription. Binding cooperativity assays indicated that protein p4 cannot interact with this mutant RNA polymerase at PA2c. Protein p4 could form a complex at PA2c with purified wild-type alpha subunit, but not with a deletion mutant lacking the 15 C-terminal residues. Our results indicate that protein p4 represses PA2c by interacting with the C-terminal domain of the alpha subunit of RNA polymerase. Therefore, this domain of the alpha subunit can receive regulatory signals not only from transcriptional activators, but from repressors also.
Monsalve
M
M
Centro de Biología Molecular Autónoma Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónomo de Madrid, Spain.
Mencía
M
M
Salas
M
M
Rojo
F
F
D017103:Bacillus Phages / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology / Q000821:virology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D008957:Models, Genetic; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D010446:Peptide Fragments; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C053987:p4 protein, Bacteriophage phi 29; D012321:DNA-Directed RNA Polymerases