During
transcription initiation by RNA polymerase II, binding of TATA box-binding protein (TBP) to promoters is a crucial rate-limiting step.
Consistent with this idea, a class of TBP mutants was identified that
causes promoter-specific transcriptional defects in vivo, resulting in
two phenotypes: inositol auxotrophy and inability to grow on media
containing galactose. In vitro, affinity of the mutant TBPs for TATA
boxes is severely reduced. We have found that co-overexpression of
TOA1 and TOA2 , genes encoding the general transcription
factor TFIIA, suppresses the phenotypes caused by the TBP mutants at the
transcriptional level. Overexpression of SUA7 , the gene encoding
TFIIB, does not suppress the mutant phenotypes. In vitro, saturating
amounts of TFIIA restore the binding of mutant TBP to the TATA box.
These findings suggest that TFIIA is important for transcriptional
activation, and the degree to which TFIIA is required by different
promoters might vary. Indeed, activation of INO1 , GAL1 and
GAL10 transcription is dramatically reduced by depletion of TFIIA
levels in vivo, while activation of other genes, such as CUP1 , is
much less affected. To investigate the function of TFIIA in vivo,
dominant mutations in TOA1 and TOA2 were identified that
suppress the mutant TBP phenotypes. According to the crystallographic
data, some of the mutations most likely cause increased affinity of
TFIIA for either DNA or TBP. Taken together, our data suggest that TFIIA
has a promoter-specific effect on transcriptional activation in vivo. At
promoters that are sensitive to TFIIA levels, TFIIA might facilitate
transcriptional activation by increasing the affinity of TBP for DNA.
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