Identification of RTF1, a Novel Gene Important for TATA Site Selection by TATA Box-Binding Protein in Saccharomyces cerevisiae

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Abstract
Interaction of the TATA box-binding protein (TBP) with promoters of RNA polymerase II-transcribed genes is an early and essential step in mRNA synthesis. Previous studies have demonstrated that the rate-limiting binding of TBP to a TATA element can be influenced by transcriptional regulatory proteins. To identify additional factors that may regulate DNA binding by TBP in vivo, we performed a genetic selection for extragenic suppressors of a yeast TBP mutant that exhibits altered and relaxed DNA binding specificity. This analysis has led to the discovery of a previously unidentified gene, RTF1. The original rtf1 suppressor mutation, which encodes a single amino acid change in Rtf1, and an rtf1 null allele suppress the effects of the TBP specificity mutant by altering transcription initiation. Differences in the patterns of transcription initiation in these strains strongly suggest that the rtf1 missense mutation is distinct from a simple loss-of-function allele. The results of genetic crosses indicate that suppression of TBP mutants by mutations in RTF1 occurs in an allele-specific fashion. In a strain containing wild-type TBP, the rtf1 null mutation suppresses the transcriptional effects of a Ty delta insertion mutation in the promoter of the HIS4 gene, a phenotype also conferred by the TBP altered-specificity mutant. Finally, as shown by indirect immunofluorescence experiments, Rtf1 is a nuclear protein. Taken together, our findings suggest that Rtf1 either directly or indirectly regulates the DNA binding properties of TBP and, consequently, the relative activities of different TATA elements in vivo.