title: Molecular mechanism of transcription inhibition by phage T7 gp2 protein.
abstract: Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β' jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β' jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the -10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β' jaw.
authors.affiliation: Department of Molecular Biology and Biochemistry, Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. mekler@waksman.rutgers.edu
authors.forename: Vladimir
authors.initials: V
authors.lastname: Mekler
authors.forename: Leonid
authors.initials: L
authors.lastname: Minakhin
authors.forename: Carol
authors.initials: C
authors.lastname: Sheppard
authors.forename: Sivaramesh
authors.initials: S
authors.lastname: Wigneshweraraj
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018983:DNA Footprinting; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D012097:Repressor Proteins; C120334:gene 2 product, bacteriophage T7; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I