title: Elucidation of the Dynamics of Transcription Elongation by RNA Polymerase II using Kinetic Network Models.
abstract: RNA polymerase II (Pol II) is an essential enzyme that catalyzes transcription with high efficiency and fidelity in eukaryotic cells. During transcription elongation, Pol II catalyzes the nucleotide addition cycle (NAC) to synthesize mRNA using DNA as the template. The transitions between the states of the NAC require conformational changes of both the protein and nucleotides. Although X-ray structures are available for most of these states, the dynamics of the transitions between states are largely unknown. Molecular dynamics (MD) simulations can predict structure-based molecular details and shed light on the mechanisms of these dynamic transitions. However, the employment of MD simulations on a macromolecule (tens to hundreds of nanoseconds) such as Pol II is challenging due to the difficulty of reaching biologically relevant timescales (tens of microseconds or even longer). For this challenge to be overcome, kinetic network models (KNMs), such as Markov State Models (MSMs), have become a popular approach to access long-timescale conformational changes using many short MD simulations. We describe here our application of KNMs to characterize the molecular mechanisms of the NAC of Pol II. First, we introduce the general background of MSMs and further explain procedures for the construction and validation of MSMs by providing some technical details. Next, we review our previous studies in which we applied MSMs to investigate the individual steps of the NAC, including translocation and pyrophosphate ion release. In particular, we describe in detail how we prepared the initial conformations of Pol II elongation complex, performed MD simulations, extracted MD conformations to construct MSMs, and further validated them. We also summarize our major findings on molecular mechanisms of Pol II elongation based on these MSMs. In addition, we have included discussions regarding various key points and challenges for applications of MSMs to systems as large as the Pol II elongation complex. Finally, to study the overall NAC, we combine the individual steps of the NAC into a five-state KNM based on a nonbranched Brownian ratchet scheme to explain the single-molecule optical tweezers experimental data. The studies complement experimental observations and provide molecular mechanisms for the transcription elongation cycle. In the long term, incorporation of sequence-dependent kinetic parameters into KNMs has great potential for identifying error-prone sequences and predicting transcription dynamics in genome-wide transcriptomes.
authors.affiliation: Department of Chemistry and State Key Laboratory of Molecular Neuroscience, Center for System Biology and Human Health, School of Science, and IAS, The Hong Kong University of Science and Technology , Kowloon, Hong Kong.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and State Key Laboratory of Molecular Neuroscience, Center for System Biology and Human Health, School of Science, and IAS, The Hong Kong University of Science and Technology , Kowloon, Hong Kong.
authors.forename: Fátima
authors.initials: F
authors.lastname: Pardo-Avila
authors.affiliation: Department of Chemistry and State Key Laboratory of Molecular Neuroscience, Center for System Biology and Human Health, School of Science, and IAS, The Hong Kong University of Science and Technology , Kowloon, Hong Kong.
authors.forename: Ilona Christy
authors.initials: IC
authors.lastname: Unarta
authors.affiliation: Department of Chemistry and State Key Laboratory of Molecular Neuroscience, Center for System Biology and Human Health, School of Science, and IAS, The Hong Kong University of Science and Technology , Kowloon, Hong Kong.
authors.forename: Peter Pak-Hang
authors.initials: PP
authors.lastname: Cheung
authors.affiliation: Department of Chemistry and State Key Laboratory of Molecular Neuroscience, Center for System Biology and Human Health, School of Science, and IAS, The Hong Kong University of Science and Technology , Kowloon, Hong Kong.
authors.forename: Guo
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Cellular and Molecular Medicine, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California , San Diego, La Jolla, California 92093, United States.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Chemistry and State Key Laboratory of Molecular Neuroscience, Center for System Biology and Human Health, School of Science, and IAS, The Hong Kong University of Science and Technology , Kowloon, Hong Kong.
authors.forename: Xuhui
authors.initials: X
authors.lastname: Huang
mesh_terms: D018360:Crystallography, X-Ray; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D012319:RNA Polymerase II / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D012319:RNA Polymerase II