RNA polymerase bound to the PR promoter of bacteriophage lambda inhibits open complex formation at the divergently transcribed PRM promoter. Implications for an indirect mechanism of transcriptional activation by lambda repressor.
We demonstrate that RNA polymerase bound at the PR promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM promoter in vitro. Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the -10 or -35 regions of PR result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM promoter. This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes. Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which PR sequences mediate PRM repression. From these assays we were able to conclude that the formation of an open complex at the PR promoter did not exclude RNA polymerase from binding at PRM. Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the PR promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRM promoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the PR promoter.
Hershberger
P A
PA
Department of Biochemistry School of Medicine, Case Western Reserve University, Cleveland, OH 44106.
deHaseth
P L
PL
D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D003851:Deoxyribonucleases / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D008957:Models, Genetic; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases