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J Biol Chem, Vol. 273, Issue 28, 17859-17864, July 10, 1998
From the Department of Biochemistry and the Center for Advanced
Molecular Biology and Immunology, School of Medicine and Biomedical
Sciences, State University of New York,
Buffalo New York 14214-3000
The general transcription factor IIB (TFIIB) is
required for accurate and efficient transcription of protein-coding
genes by RNA polymerase II (RNAPII). To define functional domains in the highly conserved N-terminal region of TFIIB, we have analyzed 14 site-directed substitution mutants of yeast TFIIB for their ability to
support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and
to form stable complexes with purified RNAPII in vitro.
Mutations impairing the formation of stable TFIIB·RNAPII complexes
mapped to the zinc ribbon fold, whereas mutations conferring downstream
shifts in transcription start site selection were identified at
multiple positions within a highly conserved homology block adjacent
and C-terminal to the zinc ribbon. These results demonstrate that the
N-terminal region of yeast TFIIB contains two separable and adjacent
functional domains involved in stable RNAPII binding and transcription
start site selection, suggesting that downstream shifts in
transcription start site selection do not result from impairment of
stable TFIIB·RNAPII binding. We discuss models for yeast start site
selection in which TFIIB may affect the ability of preinitiation
complexes to interact with downstream DNA or to affect start site
recognition by a scanning polymerase.
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