INTERACTION OF THE 82-KDA SUBUNIT OF THE VACCINIA VIRUS EARLY TRANSCRIPTION FACTOR HETERODIMER WITH THE PROMOTER CORE SEQUENCE DIRECTS DOWNSTREAM DNA-BINDING OF THE 70-KDA SUBUNIT

The vaccinia virus early transcription factor (VETF), a heterodimeric protein composed of 82- and 70-kDa subunits, interacts with viral earl y promoters at both a sequence-specific core region upstream and a seq uence-independent region downstream of the RNA start site, To determin e the VETF subunit-promoter interactions, P-32-labeled DNA targets wer e chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating t he derivatized promoter with VETF and exposing the complex to 302-nm l ight, the protein was denatured and the individual subunits with or wi thout covalently bound DNA were isolated with specific antiserum and a nalyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 d uplex probes, with uniquely positioned aryl azide moieties on the codi ng or template strands, we found that the 82-kDa subunit interacted pr imarily with the core region of the promoter, whereas the 70-kDa subun it interacted with the downstream region, Nucleotide substitutions in the core region that downregulate transcription affected the binding o f both subunits: the 82-kDa subunit no longer exhibited specificity fo r upstream regions of the promoter but also bound to downstream region s, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site, These results su ggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downst ream.