Abstract
The active sites of multisubunit RNA polymerases have a “trigger loop” (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.
Highlights
RNA polymerase II (Pol II) synthesizes all eukaryotic mRNAs
Coupling high-throughput sequencing with our previously established genetic system, we are able to assess the in vivo phenotypes for almost all possible single substitution Pol II trigger loop (TL) mutants in the budding yeast Saccharomyces cerevisiae
We show that mutants in the TL nucleotide interacting and linker regions widely confer dominant and severe growth defects
Summary
RNA polymerase II (Pol II) synthesizes all eukaryotic mRNAs. Structural studies of Saccharomyces cerevisiae (Sce) Pol II have illuminated mechanisms of transcription [1,2,3,4,5,6], especially RNA synthesis. RNA synthesis occurs through iterative nucleotide addition cycles (NACs): selection of correct substrate nucleoside triphosphate (NTP), catalysis of phosphodiester bond formation, and enzyme translocation to the template position. These critical steps in NAC appear to be coordinated by a critical, conserved domain within the Pol II active site: the trigger loop (TL). In a simplified two-step model, correct NTP binding appears to facilitate TL movement such that a bound, matched NTP shifts the TL from the “open” state to the “closed” state [4,15,16,17,18], allowing capture of the matched NTP in the Pol II active site and promotion of phosphodiester bond formation [4,17,19]. Distinct TL conformations or interactions are linked to different functions in transcription, with delicate control of TL dynamics promoting proper transcription elongation while possibly incorporating signals from the rest of Pol II or Pol II bound factors [17,33,34,35,36]
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