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J. Biol. Chem., Vol. 266, Issue 23, 15539-15544, 08, 1991

Promoter DNA contacts made by the vaccinia virus early transcription factor

SS Broyles, J Li and B Moss
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.

Vaccinia virus RNA polymerase requires the heterodimeric protein, vaccinia early transcription factor (VETF), for transcription of early gene templates in vitro. We have analyzed the vaccinia growth factor promoter sequences interacting with VETF at the nucleotide level and provide evidence that the factor contacts the DNA at two separate sites. DNase I protection analysis showed that VETF was found to nucleotides -12 to -29 relative to the transcription initiation site, and also to nucleotides +8 to +10 downstream of the initiation site. The importance of both binding sites for stable complex formation was supported by methylation interference analysis. Using synthetic oligonucleotides encoding different parts of the vaccinia growth factor promoter, it was shown that nucleotides down-stream of the transcription initiation site are required for stable complex formation. Competition binding experiments demonstrated that only the upstream binding site contributes significantly to binding specificity. Binding to two separated DNA sequences results in a bend in the promoter DNA as demonstrated by electrophoretic mobility shift analysis of permuted DNA fragments. These findings suggest that VETF activates transcription by sequence specific binding and structural alteration of the promoter DNA helix.
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