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Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser.
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.
Afiliación
  • Lin G; Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
  • Barnes CO; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
  • Weiss S; Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
  • Dutagaci B; Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824.
  • Qiu C; Department of Genetics, Harvard Medical School, Boston, MA 02115.
  • Feig M; Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824.
  • Song J; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
  • Lyubimov A; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
  • Cohen AE; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
  • Kaplan CD; Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260.
  • Calero G; Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
Proc Natl Acad Sci U S A ; 121(36): e2318527121, 2024 Sep 03.
Article en En | MEDLINE | ID: mdl-39190355
ABSTRACT
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transcripción Genética / ARN Polimerasa II / Dominio Catalítico / Simulación de Dinámica Molecular / Magnesio Idioma: En Revista: Proc Natl Acad Sci U S A Año: 2024 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transcripción Genética / ARN Polimerasa II / Dominio Catalítico / Simulación de Dinámica Molecular / Magnesio Idioma: En Revista: Proc Natl Acad Sci U S A Año: 2024 Tipo del documento: Article Pais de publicación: Estados Unidos