title: Recruitment of vaccinia virus RNA polymerase to an early gene promoter by the viral early transcription factor.
abstract: Transcription of vaccinia virus early genes in vitro requires the virally encoded RNA polymerase and early transcription factor, VETF. VETF is a promoter-binding protein with DNA-dependent ATPase activity. We have investigated the functional role of VETF in transcription activation by analyzing the interaction between the RNA polymerase and promoter DNA. Using a gel shift assay, a novel protein-DNA complex was detected that required both RNA polymerase and VETF. The complex was suggested to be a transcription initiation complex by its ability to incorporate 32P-labeled nucleotides in combinations compatible with synthesis of a short RNA chain. Competition binding studies indicated that the RNA polymerase associated specifically with a viral early promoter. These experiments demonstrate that VETF activates transcription by directly recruiting the RNA polymerase to the promoter. Sedimentation analysis showed that VETF and RNA polymerase did not form a stable complex unless promoter DNA was present, indicating that protein-protein contacts are not the sole basis for initiation complex assembly. DNase I cleavage and methylation interference analyses revealed a hyperreactive site in the center of the promoter. Radiolabeling of RNA in the RNA polymerase-promoter complex did not occur when AMP-PNP (adenyl-5'-yl imidodiphosphate) was substituted for ATP, suggesting that ATP hydrolysis is required for the initiation of transcription. A model is proposed to account for these findings.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: S S
authors.initials: SS
authors.lastname: Broyles
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000378:metabolism* / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D014764:Viral Proteins; C064449:early transcription factor protein, Vaccinia virus; D012321:DNA-Directed RNA Polymerases; D000251:Adenosine Triphosphatases