We wish to understand how eukaryotic genes are expressed.
The RNA polymerase II transcription complex contains several dozen
proteins, but only a few of these have well defined functions. Using a
combination of genetic and biochemical assays, we are attempting to
determine the role each protein plays in transcription initiation, as
well as how mRNA processing is specified to transcripts produced by pol
II. TFIID binds to the basal promoter, providing a platform for binding
by polymerase and other factors. We find that TFIID contains a
subcomplex that resembles the histone octamer of the nucleosome. In
contrast to other TFIID subunits, the histone-like subunits appear to be
generally required for transcription. TFIIB bridges the promoter-bound
TFIID complex and pol II. We have characterized a TFIIB mutant that
shifts the initation site of transcription. In vitro, this mutant can
assemble into transcription complexes normally, but is transcriptionally
inactive. Therefore, TFIIB must also function after transcription
complex assembly, perhaps mediating a conformation change that brings
the active site of polymerase to the initiation site. Coincident with
the initiation of transcription, the CTD of pol II is extensively
phosphorylated by the TFIIH-associated kinase. We and others have found
that this phosphorylated domain acts as a binding site for the mRNA
capping enzyme and other RNA processing enzymes, thereby coupling
transcription with mRNA processing.
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