title: Kinetics of nucleotide entry into RNA polymerase active site provides mechanism for efficiency and fidelity.
abstract: During transcription, RNA polymerase II elongates RNA by adding nucleotide triphosphates (NTPs) complementary to a DNA template. Structural studies have suggested that NTPs enter and exit the active site via the narrow secondary pore but details have remained unclear. A kinetic model is presented that integrates molecular dynamics simulations with experimental data. Previous simulations of trigger loop dynamics and the dynamics of matched and mismatched NTPs in and near the active site were combined with new simulations describing NTP exit from the active site via the secondary pore. Markov state analysis was applied to identify major states and estimate kinetic rates for transitions between those states. The kinetic model predicts elongation and misincorporation rates in close agreement with experiment and provides mechanistic hypotheses for how NTP entry and exit via the secondary pore is feasible and a key feature for achieving high elongation and low misincorporation rates during RNA elongation.
authors.affiliation: Department of Biochemistry & Molecular Biology, 603 Wilson Rd., Room 218 BCH, Michigan State University, East Lansing, MI 48824, USA. Electronic address: bbwang@msu.edu.
authors.forename: Beibei
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Biochemistry & Molecular Biology, 603 Wilson Rd., Room 218 BCH, Michigan State University, East Lansing, MI 48824, USA. Electronic address: sextonra@msu.edu.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Sexton
authors.affiliation: Department of Biochemistry & Molecular Biology, 603 Wilson Rd., Room 218 BCH, Michigan State University, East Lansing, MI 48824, USA. Electronic address: feig@msu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Feig
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009711:Nucleotides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D055672:Static Electricity
chemical_list: D009711:Nucleotides; D012321:DNA-Directed RNA Polymerases
keywords: Markov state model; Molecular dynamics simulation; NTP discrimination; Network model; Secondary pore