The DNA-dependent ATPase activity of vaccinia virus early gene transcription factor is essential for its transcription activation function.
Vaccinia virus early transcription factor (VETF) activates the transcription of early gene templates by the viral RNA polymerase. VETF is a heterodimeric protein that binds to transcription promoters and has an associated DNA-dependent ATPase activity. The small subunit of VETF has sequences resembling two motifs commonly found in ATPases: an A-type ATP binding motif and a DEAH box. To investigate the functional role of the ATPase activity, we have analyzed the effect of mutations in each of the putative ATPase motifs. Recombinant VETF was expressed in HeLa cells using a vaccinia virus/T7 RNA polymerase system. Simultaneous expression of both subunits of VETF was required to obtain soluble protein with promoter binding, DNA-dependent ATPase, and transcription activation functions. The mutants with altered ATPase motifs retained promoter binding activity but had no detectable ATPase activity and no ability to activate transcription. The DEAH box mutant was shown to dominantly repress transcription activation by wildtype VETF. These results indicate that the DNA-dependent ATPase activity of VETF is essential for its transcription activation function.
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
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Broyles
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D004265:DNA Helicases; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; C064449:early transcription factor protein, Vaccinia virus; D000251:Adenosine Triphosphatases; D004265:DNA Helicases