Regulation of the TATA -binding protein by a general transcription factor, NC2

Abstract

The TATA-binding protein (TBP) performs essential roles in transcription by all three nuclear RNA polymerases. Several proteins have been proposed to repress RNA polymerase II transcription by directly inhibiting TBP activity, yet there was little supporting evidence from in vivo studies. I isolated three groups of mutations in yeast TBP that, unlike most reported TBP mutations with decreased transcriptional activity, increase transcription from core promoters. The majority of the mutated residues, constituting one of the three groups, form a tight cluster on the exposed surface near the TFIIB binding sites though residing in four different sub-domains of TBP. This new TBP surface region interacts both genetically and physically with a proposed general transcription repressor, Negative Co-factor 2 (NC2). The mutant TBPs in this group are resistant to NC2 repression, thus allowing the binding of TFIIA and TFIIB to TBP and the subsequent transcription initiation. The other two groups of mutants are defective for Mot1 binding and TBP self-dimerization respectively, which are likely to contribute to the transcriptional hyperactivities of the mutants. It is further shown that multiple repressors possess overlapping functions to keep basal promoter elements inactive under appropriate conditions.;An NC2 mutant and one of the TBP mutants defective for binding NC2 were subjected to genome-wide analysis, which revealed a potential positive role of NC2 in transcription of many genes. It was unknown, however, whether that positive role was direct or what underlay its molecular mechanism. I demonstrated that NC2 has dual direct roles in vivo and in vitro, repressing basal transcription and stimulating activated transcription in a promoter-specific manner. Further equilibrium studies showed that NC2 could form a complex with TBP-DNA that is much more stable than TBP-DNA alone. Based on these results, a new two-step model for regulation of TBP by NC2 was proposed that challenges the current simplified view of the function of this transcriptional regulator.