Biochemical interplay between subunits of yeast RNA polymerase II transcription machinery

Transcription of nuclear protein-encoding genes by eukaryotic RNA polymerase II (pol II) requires cis-acting promoter DNA sequences and at least five initiation factors or general transcription factors, designated TFIIB, TFIID, TFIIE, TFIIF and TFIIH. Initiation factors assemble on promoter DNA and recruit pol II to form a stable preinitiation complex that can initiate transcription in vitro upon addition of ribonucleotide substrates for RNA synthesis. The yeast preinitiation complex contains at least 27 distinct polypeptide chains (12 polymerase subunits and 15 subunits comprising the five initiation factors). These proteins must function in a concerted manner to ensure proper regulation of gene expression for normal cell growth and development.;Using a well-defined yeast reconstituted system to explore the molecular and biochemical mechanisms of transcription initiation and its regulation, we have discovered novel functional interactions between RNA polymerase II subunits and the initiation factors. We find that a previously uncharacterized single-stranded (ss)-DNA binding domain in the Rpb7 polymerase subunit is essential for promoter utilization and that its function comes into play after promoter binding by the polymerase. We also find that although the evolutionarily conserved C-terminal repeat domain (CTD) in the Rpb1 polymerase subunit is entirely dispensable for transcription in vitro, CTD phosphorylation by the TFIIH-associated protein kinase is strongly activated by DNA and the other initation factors. We also identify the core TFIIH subunits that mediate CTD kinase activation. Implications for the essential role of the TFIIH CTD kinase in cellular mRNA synthesis are discussed.