Structural and Functional Analysis of Mutations along the Crystallographic Dimer Interface of the Yeast TATA Binding Protein

Abstract

The TATA binding protein (TBP) is a central component of the eukaryotic transcription machinery and is subjected to both positive and negative regulation. As is evident from structural and functional studies, TBP's concave DNA binding surface is inhibited by a number of potential mechanisms, including homodimerization and binding to the TAND domain of the TFIID subunit TAF1 (yTAF_II145/130). Here we further characterized these interactions by creating mutations at 24 amino acids within the Saccharomyces cerevisiae TBP crystallographic dimer interface. These mutants are impaired for dimerization, TAF1 TAND binding, and TATA binding to an extent that is consistent with the crystal or nuclear magnetic resonance structure of these or related interactions. In vivo, these mutants displayed a variety of phenotypes, the severity of which correlated with relative dimer instability in vitro. The phenotypes included a low steady-state level of the mutant TBP, transcriptional derepression, dominant slow growth (partial toxicity), and synthetic toxicity in combination with a deletion of the TAF1 TAND domain. These phenotypes cannot be accounted for by defective interactions with other known TBP inhibitors and likely reflect defects in TBP dimerization.

ACKNOWLEDGMENTS

We thank Lata Chitikila, Diane Alexander, and Kevin Halbe for technical assistance and members of the Pugh laboratory for helpful discussions.

This work was supported by NIH grant GM59055.