title: Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution.
abstract: RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous 'synthetic nucleic acid substitution' strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences the rate of P-O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing β,γ-bridging atom-'substituted' NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and non-cognate nucleotide incorporation. Intriguingly, the effect of the β,γ-methylene substitution on the non-cognate UTP/dT scaffold (∼3-fold decrease in kpol) is significantly different from that of the cognate ATP/dT scaffold (∼130-fold decrease in kpol). Removal of the wobble hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP. Our kinetic and modeling studies are consistent with a unique altered transition state for bond formation and cleavage for UTP/dT incorporation compared with ATP/dT incorporation. Collectively, our data reveals the functional interplay between NTP triphosphate moiety and base pair hydrogen bonding recognition during nucleotide incorporation.
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA.
authors.forename: Candy S
authors.initials: CS
authors.lastname: Hwang
authors.affiliation: Department of Cellular and Molecular Medicine, School of Medicine; Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Cellular and Molecular Medicine, School of Medicine; Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5017, USA Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS, Université Montpellier, ENSCM, Ecole Nationale Supérieure de Chimie de Montpellier, 8 Rue de l'Ecole Normale, 34296 Montpellier cedex 5, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Ulrich
authors.affiliation: Department of Chemistry, Division of Biomedical Engineering, Center of Systems Biology and Human Health, School of Science and Institute for Advance Study, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Cellular and Molecular Medicine, School of Medicine; Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA.
authors.forename: Jenny
authors.initials: J
authors.lastname: Chong
authors.affiliation: Department of Cellular and Molecular Medicine, School of Medicine; Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA.
authors.forename: Ji Hyun
authors.initials: JH
authors.lastname: Shin
authors.affiliation: Department of Chemistry, Division of Biomedical Engineering, Center of Systems Biology and Human Health, School of Science and Institute for Advance Study, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
authors.forename: Xuhui
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5017, USA.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA mckenna@usc.edu.
authors.forename: Charles E
authors.initials: CE
authors.lastname: McKenna
authors.affiliation: Department of Cellular and Molecular Medicine, School of Medicine; Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA dongwang@ucsd.edu.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
mesh_terms: D020029:Base Pairing; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000378:metabolism
chemical_list: D009711:Nucleotides; D012313:RNA; D014542:Uridine Monophosphate; D012319:RNA Polymerase II