title: Five checkpoints maintaining the fidelity of transcription by RNA polymerases in structural and energetic details.
abstract: Transcriptional fidelity, which prevents the misincorporation of incorrect nucleoside monophosphates in RNA, is essential for life. Results from molecular dynamics (MD) simulations of eukaryotic RNA polymerase (RNAP) II and bacterial RNAP with experimental data suggest that fidelity may involve as many as five checkpoints. Using MD simulations, the effects of different active site NTPs in both open and closed trigger loop (TL) structures of RNAPs are compared. Unfavorable initial binding of mismatched substrates in the active site with an open TL is proposed to be the first fidelity checkpoint. The leaving of an incorrect substrate is much easier than a correct one energetically from the umbrella sampling simulations. Then, the closing motion of the TL, required for catalysis, is hindered by the presence of mismatched NTPs. Mismatched NTPs also lead to conformational changes in the active site, which perturb the coordination of magnesium ions and likely affect the ability to proceed with catalysis. This step appears to be the most important checkpoint for deoxy-NTP discrimination. Finally, structural perturbations in the template DNA and the nascent RNA in the presence of mismatches likely hinder nucleotide addition and provide the structural foundation for backtracking followed by removing erroneously incorporated nucleotides during proofreading.
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Beibei
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Kristopher
authors.initials: K
authors.lastname: Opron
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Zachary F
authors.initials: ZF
authors.lastname: Burton
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Robert I
authors.initials: RI
authors.lastname: Cukier
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA feig@msu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Feig
mesh_terms: D020029:Base Pairing; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D012319:RNA Polymerase II / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013698:Templates, Genetic; D016963:Thermus thermophilus / Q000201:enzymology; D014158:Transcription, Genetic
chemical_list: D012313:RNA; D004247:DNA; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases