title: Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes.
abstract: The RNA polymerase II (Pol II) is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the "secondary channel" is the only route for NTP to reach the active site of the enzyme or if the "main channel" could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB) is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP) substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD) simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription.
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: Daniel-Adriano
authors.initials: DA
authors.lastname: Silva
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: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, United States of America.
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: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000648:ultrastructure*; D004058:Diffusion; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry* / Q000648:ultrastructure*; D012319:RNA Polymerase II / Q000737:chemistry* / Q000648:ultrastructure*; D014158:Transcription, Genetic
chemical_list: D009711:Nucleotides; D012313:RNA; D004247:DNA; D012319:RNA Polymerase II