Initiation of class II gene expression in eukaryotes involves the formation of a large multisubunit complex containing RNA polymerase II and additional factors on promoters. TATA-binding protein (TBP) plays a central role in this process as it binds directly to promoter DNA. At least five different TBP-associated protein complexes have been identified that have roles in class II gene transcription, but their contributions to the regulation of gene expression are not yet well understood These TBP-binding proteins include TAFIIs, NC2 (Dr1-DRAP1), MOT1, NOTs and SPTs. The genetic and biochemical experiments described in this proposal are designed to investigate the nature and function of TBP-binding proteins other than TAFIIs in control of class II gene expression in S. cerevisiae. The four specific aims of this proposal are: 1) to study the genetic interaction betwee RNA polymerase II holoenzyme and TBP-binding proteins in vivo, 2) to investigate the effects of mutations in the TBP-binding proteins on class II gene expression in vivo, 3) to purify and identify the subunit composition of the various TBP-binding proteins involved in class II gene expression in S. cerevisiae using biochemical approaches, 4) to study the transcriptional functions of the TBP-binding proteins using wild-type and mutant proteins in transcription systems in vitro.
