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High-resolution phenotypic landscape of the RNA Polymerase II trigger loop

By Chenxi Qiu, Olivia C. Erinne, Jui Dave, Ping Cui, Huiyan Jin, Nandhini Muthukrishnan, Leung K. Tang, Sabareesh Ganesh Babu, Kenny C. Lam, Paul J. Vandeventer, Ralf Strohner, Jan Van den Brulle, Sing-Hoi Sze, Craig D. Kaplan

Posted 14 Aug 2016
bioRxiv DOI: 10.1101/068726 (published DOI: 10.1371/journal.pgen.1006321)

The active site of multicellular 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 major 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.

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