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 early promoters at both a sequence-specific core region upstream and a sequence-independent region downstream of the RNA start site. To determine the VETF subunit-promoter interactions, 32P-labeled DNA targets were chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating the derivatized promoter with VETF and exposing the complex to 302-nm light, the protein was denatured and the individual subunits with or without covalently bound DNA were isolated with specific antiserum and analyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 duplex probes, with uniquely positioned aryl azide moieties on the coding or template strands, we found that the 82-kDa subunit interacted primarily with the core region of the promoter, whereas the 70-kDa subunit interacted with the downstream region. Nucleotide substitutions in the core region that downregulate transcription affected the binding of both subunits: the 82-kDa subunit no longer exhibited specificity for upstream regions of the promoter but also bound to downstream regions, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site. These results suggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downstream.
Cassetti
M A
MA
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0455, USA.
Moss
B
B
D001386:Azides; D001483:Base Sequence; D018983:DNA Footprinting; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013873:Thionucleotides; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D001386:Azides; D004279:DNA, Viral; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides; D014157:Transcription Factors; D014764:Viral Proteins; C064449:early transcription factor protein, Vaccinia virus; C011171:4-azidophenacyl bromide