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

Proc Natl Acad Sci U S A. 2024 Sep 3;121(36):e2318527121. doi: 10.1073/pnas.2318527121. Epub 2024 Aug 27.

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.

Keywords: RNA polymerase II; XFEL; catalytic mechanisms; magnesium chemistry; transcription.

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism
  • Binding Sites
  • Catalytic Domain*
  • Electrons
  • Lasers
  • Magnesium* / chemistry
  • Magnesium* / metabolism
  • Molecular Dynamics Simulation*
  • Protein Binding
  • Protein Conformation
  • RNA Polymerase II* / chemistry
  • RNA Polymerase II* / genetics
  • RNA Polymerase II* / metabolism
  • Transcription, Genetic*

Substances

  • RNA Polymerase II
  • Magnesium
  • Adenosine Triphosphate