Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free- electron laser

• 成果类型: Article
• 署名作者: Lin, Guowu; Barnes, Christopher O.; Weiss, Simon; Dutagaci, Bercem; Qiu, Chenxi; Feig, Michael; Song, Jihnu; Lyubimov, Artem; Cohen, Aina E.; Kaplan, Craig D.; Calero, Guillermo
• 署名单位: Pennsylvania Commonwealth System of Higher Education (PCSHE); University of Pittsburgh; California Institute of Technology; Michigan State University; Harvard University; Harvard Medical School; Stanford University; United States Department of Energy (DOE); SLAC National Accelerator Laboratory; Pennsylvania Commonwealth System of Higher Education (PCSHE); University of Pittsburgh; Stanford University; University of California System; University of California Merced
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8827
• DOI: 10.1073/pnas.231852712
• 发表日期: 2024-09-03
• 关键词: trigger-loop; crystal-structure; elongation complex; acid polymerases; FIDELITY; dna; mechanism; crystallography; mutations; DYNAMICS

摘要

Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and, more importantly, a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.