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
Mc. Cassetti et B. Moss, 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, Proceedings of the National Academy of Sciences of the United Statesof America, 93(15), 1996, pp. 7540-7545
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.