BPS2026 – TFIIS enhancement of Pol II cleavage is kinetically controlled

The eukaryotic RNA polymerases (Pols) contain subunits or interact with trans-acting factors that confer robust endonuclease activity. A12.2 and C11 are subunits of Pol I and Pol III, respectively. Pol II, however, recruits transcription factor IIS (TFIIS). This evolutionary divergence of A12.2 and C11 being subunits while TFIIS is a trans-acting factor suggests functional divergence of these domains. Our studies aim to uncover the mechanistic impact of TFIIS on Pol II-catalyzed nucleotide incorporation and endonuclease activity. Single turnover in vitro transcription assays were performed with Saccharomyces cerevisiae TFIIS and Pol II. Nucleotide incorporation time courses were collected as a function of both TFIIS and nucleotide concentrations. Global non-linear least-squares analysis of these time courses revealed that TFIIS binds to elongation complexes after nucleotide incorporation because TFIIS binding is slow relative to correct nucleotide incorporation. However, if subsequent nucleotide incorporation is slow, TFIIS has adequate time to bind and activate Pol II’s endonuclease activity. Together, these observations suggest that TFIIS binds to Pol II elongation complexes once they have stalled. Thus, we hypothesize that the mechanism of TFIIS-stimulated “proofreading” is kinetically controlled. The implications of these findings on the evolutionary divergence of the eukaryotic Pols will be discussed.