In RNA
polymerase II transcription, TBP functions in a multi-subunit complex
(TFIID) that consists of TBP and TBP associated factors (TAFs). TBP is
also required for transcription by Pol I and Pol III. Here, we present
the initial characterization of a yeast temperature-sensitive TBP mutant
(K151L; K156Y) that is specifically defective for Pol II transcription.
This TBP mutant is functional for both Pol I and Pol III transcription,
and also appears to be functional for basal and activated transcription
from Pol II promoters containing a TATA element. In contrast, this
allele may be defective for transcriptional activity at promoters
lacking a canonical TATA element. Currently it is not known whether
there is a difference between the mechanism of transcription at TATA-containing and TATA-less promoters. To determine the specificity of this
defect we are expanding our studies to include newly designed probes for
other TATA-less promoters. Initial biochemical data indicates that this
TBP mutant does not remain part of an intact TFIID complex at the non-permissive temperature (the mutant TFIID is severely depleted for
various TAFs). An attractive hypothesis that is also supported by work
from other laboratories is that certain TAFs are more critical at TATA-less promoters. Using site-directed mutagenesis we have created single
mutants at amino acid 151 or 156 and found that both mutations are
required for the lethal phenotype at the non-permissive temperature.
Future studies may allow us to more clearly define the role of TBP in
transcription by Pol II at TATA-less promoters.
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