Mutations on the DNA Binding Surface of TBP Discriminate between Yeast TATA and TATA-Less Gene Transcription

Abstract

Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription.

ACKNOWLEDGMENTS

We thank B. Knutson and J. Fishburn for help with RNA analysis, protein expression, protein purification, and in vitro transcription assays, S. Buratowski for the TAF11 ts strain, P. A. Weil's laboratory for advice on preparing whole-cell extracts, F. Pugh and H. S. Rhee for sharing their ChIP-exo data and for discussions, J. Geiger for discussions on TBP binding mechanisms, and B. Knutson and S. Grünberg for comments on the manuscript.

This work was supported by grant 2RO1GM053451 from the NIH to S.H.