Abstract
The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5′ and 3′ parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.
We thank Andy Sharrocks, Paul Shore, and members of the lab for comments on the manuscript and also members of the Division of Gene Expression at Dundee University for helpful comments during this study. We are also grateful to Ben Luisi and Tali Haran for many discussions and critiques of the data.
R.E. was supported by a research studentship from the MRC. N.A.H. was the recipient of a BBSRC research studentship. This work was funded by the BBSRC and the Wellcome Trust (061207/Z/00/Z/CH/TG/dr). S.G.E.R. is a Wellcome Trust Senior Research Fellow.