MECHANISTIC INSIGHTS INTO THE FUNCTION OF SSL2/TFIIH IN RNA POLYMERASE II TRANSCRIPTION START SITE SCANNING

The initial step of RNA polymerase II (Pol II) transcription involves a large number of transcription factors and arises at multiple sites within most promoters. TFIIH is an essential, multisubunit transcription factor that assembles on promoter DNA with Pol II and five other general transcription factors (GTFs) TFIIA, TFIIB, TFIID, TFIIE and TFIIF to form a pre-initiation complex (PIC) for basal transcription. During transcription initiation, TFIIH melts promoter DNA through the ATPase activity of its Ssl2 subunit. In the model eukaryote Saccharomyces cerevisiae, after DNA melting, Pol II scans downstream for usable transcription start sites (TSSs). TFIIH has been proposed as responsible for promoter scanning steps downstream of promoter melting but this has not been tested extensively. ssl2 mutations affect TSS selection, consistent with Ssl2-dependent functions of TFIIH in scanning. We hypothesize that TFIIH serves as the engine for scanning by affecting TSS usage in at least two possible ways: by controlling the processivity (how far) of scanning, and/or the rate at which scanning translocates (how fast). To understand the function of Ssl2/TFIIH in promoter scanning and TSS selection, we identified novel alleles of SSL2 in genetic screens, mutants defective in TSS distribution that may potentially arise from altered scanning. Consistent with this notion, these ssl2 alleles alter scanning in ways that are distinct from how changes to the Pol II active site alter scanning and this difference is observed genome-wide. To understand further how Ssl2/TFIIH and Pol II or other initiation factors work concurrently to promote transcription initiation, we performed genetic interaction experiments between initiation factors. Our genetic data indicate that there are at least two major networks controlling promoter scanning and TSS selection, one controls the efficiency of initiation through Pol II activity or factors regulating Pol II’s activity; another network hypothetically controls the processivity of scanning by TFIIH. Moreover, we are examining if promoter scanning is a conserved mechanism across eukaryotes. We are asking if perturbation of transcription initiation in Schizosaccharomyces pombe and Drosophila melanogaster through mutation of initiation factors fits expectations of a scanning mechanism. Our preliminary data in both organisms indicate differences from S. cerevisiae.