| dc.description.abstract | mRNA synthesis by RNA polymerase II (Pol II) is an essential process in
eukaryotes. In my dissertation, I have undertaken two parallel approaches to expand our
understanding of mechanism of Pol II, a large twelve-subunit protein complex in
budding yeast Saccharomyces cerevisiae. First, we develop a high-throughput genetic
platform to dissect functions of every residue in a critical Pol II active site domain: the
trigger loop (TL). The TL multitasks in catalysis and translocation through its distinct
conformational states, alteration of which causes wide-ranging transcription defects in
vitro and in vivo. By establishing the correlation between a set of in vivo conditional
growth phenotypes and in vitro biochemical defects, our genetic data allows us to predict
biochemical defects and alteration of TL states in nearly all TL single substitution
variants. For example, we provide evidence supporting critical contribution of an intra-
TL hydrophobic pocket in stabilizing the off-catalytic TL state, as evidenced by
mutations disrupting the pocket confer phenotypes consistent with increased catalysis
and infidelity. These data are also consistent with a critical role of this intra-TL pocket in
promoting Pol II fidelity. In addition, we show diverse allele-specific genetic
interactions among TL and TL surrounding domains, supporting possible contribution of
the TL surrounding funnel and bridge helices to TL dynamics and function. Second, we
characterize the mode of action of thiolutin, a well-known transcription inhibitor with
unclear mechanism of transcription inhibition. Recent studies demonstrated that thiolutin
inhibited multiple metalloproteins through Zn^2+ chelation, but failed to observe direct
thiolutin inhibition of purified RNA polymerases, suggesting additional factors are
needed for thiolutin-mediated transcription inhibition or that the inhibition is indirect.
We have taken chemical genetics and biochemical approaches to investigate the thiolutin
mode of action. While characterizing multiple thiolutin effects in vivo, we demonstrate
that thiolutin, when activated by DTT and Mn^2+, directly inhibits Pol II in vitro. We
further investigate the nature of the inhibitory species and the property of the inhibited
Pol II. We suggest that thiolutin inhibits Pol II through a novel mechanism distinct from
most other known RNA polymerase inhibitors. Taken together, we develop a highthroughput
phenotypic system to dissect functions of Pol II TL residues and characterize
a novel mode of action of thiolutin. | en |
