In an effort to identify
proteins that have general roles in transcription by RNA polymerase II,
we have previously selected for mutations that cause increased
transcription from suc2deltauas (a transcriptionally inactive
allele of SUC2 ). Our original selection identified recessive
mutations in six BUR genes ( BUR = Bypass UAS Requirement)
that encode general repressors of transcription. Bur3 (=Mot1) and Bur6
repress transcription by directly inhibiting the TATA-binding protein
(TBP), while the other Bur proteins have been proposed to inhibit
transcription via chromatin-mediated repression. Here we describe a new
class of bur mutations. We have mutagenized the gene that encodes
yeast TBP ( SPT15 ), selecting for TBP mutations that increase
transcription from suc2deltauas . TBP alleles arising from this
selection were expected to be UAS-independent either due to loss of
repression or due to an increase of an intrinsic TBP activity. The
majority of our TBP mutations (34 out of 36) map to a very tight cluster
of residues on the exposed surface of TBP. This cluster is likely to
define a previously unidentified interface for contact with a repressor
of TBP. In addition to these clustered mutations, we have also
identified two other Bur - TBP surface mutations: the first
shows genetic interactions with MOT1 , while the second mutation
lies on the DNA-binding surface of TBP. We will present further genetic
and biochemical analysis of these hyperactive TBP alleles.
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