The role of TFIIB, XPB and TFIIE in transcription initiation in Schizosaccharomyces pombe

Eukaryotic transcription by RNA polymerase II typically requires general transcription factors (GTFs) TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH binding to the core promoter elements to form a pre-initiation complex (PIC). The formation and stability of the PIC depend on the protein-DNA and protein-protein interactions of these components. When the PIC is complete, GTFs can change the conformation of the PIC to allow the polymerase to synthesize mRNA, escape from the promoter and enter the elongation mode.; GTFs can play different roles to regulate gene expression at different steps of transcription initiation. In this thesis transcription factors TFIIB, the XPB subunit of TFIIH and TFIIE were studied biochemically in S. pombe to learn how these factors work in transcription. The results show that the transcription machinery for start site selection in the fission yeast S. pombe is more mammalian-like than the budding yeast S. cerevisiae. Exogenously expressed human TFIIB can be incorporated into the S. pombe system and shift the start site of S. pombe to the closer edge of TATA whereas S. cerevisiae TFIIB cannot do this. By swapping different regions of TFIIB we suggest that the C-terminus of TFIIB is critical for choosing the mammalian start sites. This differs from the proposed properties of S. cerevisiae TFIIB.; In addition, promoter melting in S. pombe is also mammalian-like as its transcription bubble is ∼20 base pairs in size and also ATP-dependent. The ATPase and helicase motifs of XPB are also used to regulate promoter melting. TFIIB can shift the start site but it does not affect promoter melting. Therefore although the regions for promoter melting and start site selection overlap, these processes are likely independent and controlled by different factors.; Besides XPB, TFIIE is also involved in promoter melting but how it works is not clear. Our results show that TFIIEbeta contains two partner switching regions. XPB binds one of these in the leucine-rich region and disrupts TFIIEbeta homodimers. In the bHL region of TFIIEbeta, the single-stranded DNA created by promoter melting can be switched in and TFIIB is switched out. This relay switching mechanism makes TFIIEbeta a central player to regulate promoter opening and escape in transcription initiation and the transition from initiation to elongation.