Promoter-Specific Shifts in Transcription Initiation Conferred by Yeast TFIIB Mutations Are Determined by the Sequence in the Immediate Vicinity of the Start Sites

Abstract

The general transcription factor IIB (TFIIB) is required for transcription of class II genes by RNA polymerase II. Previous studies demonstrated that mutations in the Saccharomyces cerevisiae SUA7 gene, which encodes TFIIB, can alter transcription initiation patterns in vivo. To further delineate the functional domain and residues of TFIIB involved in transcription start site utilization, a genetic selection was used to isolate S. cerevisiae TFIIB mutants exhibiting downstream shifts in transcription initiation in vivo. Both dominant and recessive mutations conferring downstream shifts were identified at multiple positions within a highly conserved homology block in the N-terminal region of the protein. The TFIIB mutations conferred downstream shifts in transcription initiation at the ADH1 and CYC1 promoters, whereas no significant shifts were observed at the HIS3 promoter. Analysis of a series of ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions revealed that the feature that renders a promoter sensitive to TFIIB mutations is the sequence in the immediate vicinity of the normal start sites. We discuss these results in light of possible models for the mechanism of start site utilization by S. cerevisiae RNA polymerase II and the role played by TFIIB.

ACKNOWLEDGMENTS

We thank Michael Hampsey for CYC1 plasmids, Lynne Pajak and Tom Smith for technical assistance, and Michelle Ammerman, Tim Pardee, and Lynn Ziegler for helpful discussions.

This work was supported by a grant from National Science Foundation (MCB-9905418) and a Public Health Service grant from the National Institutes of Health (GM51124) to A.S.P.