Recruitment of vaccinia virus RNA polymerase to an early gene promoter by the viral early transcription factor.
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.
Li
J
J
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Broyles
S S
SS
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*
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