In Vivo Consequences of Altered Pol II Catalysis

Abstract

Gene transcription by RNA polymerase II (Pol II) is an essential process. Using Saccharomyces cerevisiae as a model system, our lab has previously identified and partially characterized Pol II activity mutants that can alter catalysis rate to faster or slower than wild type in vitro. In my dissertation research I use a set of these Pol II activity mutants to determine consequences of altered catalysis rate on polymerase functions, co-transcriptional pre-mRNA processing and gene expression in vivo. I show that alteration in Pol II catalytic rate, either increase or decrease, leads to a decreased Pol II occupancy and an apparent reduction in elongation rate on a commonly used reporter gene in vivo. Measurement of in vivo elongation rate on this reporter requires transcriptional shutoff followed by ChIP. I discover that some Pol II catalytic mutants can compromise the kinetics of transcription shutoff by glucose, which is generally assumed to be unaffected by transcription mutants. Further, I show that Pol II catalytic mutants affect model gene expression and the effects on gene expression are exacerbated with increased promoter strength and gene length. My results suggest that gene expression defects in the Pol II mutants may in part result from defective mRNA processing. Additionally, I show that mRNA half-lives for that model gene are increased in Pol II mutant strains and the magnitude of half-life changes correlate both with mutants’ growth and the magnitude of reporter gene expression defects. Finally, I test if altered Pol II elongation sensitizes cells to nucleotide depletion and find that Pol II mutants and several elongation factor mutants respond to GTP starvation similarly to wild type and that putative elongation defects are not likely to drive the cellular response to limiting GTP. Altogether my findings reveal wide-ranging in vivo effects of Pol II catalytic mutants, which will be critical for precise use of these Pol II catalytic mutants in gene regulation studies.