Exploration Of Kinetics Of RNA Polymerase Ⅱ Binding To Substrate Molecules By Coarse-grained Molecular Dynamics Simulations

RNA polymerase Ⅱ is the central machine of the transcription process in eukaryotic cells.RNA polymerase II synthesizes nucleotide triphosphates(NTPs)complementary to the DNA template to the RNA end to elongate the RNA chain,and its fidelity is mainly guaranteed by the selection of the ligand NTP.Initial studies indicated that RNA polymerase II may have two channels: the main channel and the secondary channel.More experimental evidence indicated that the secondary channel was the main pathway for ligand entry and exit.However,current experimental methods are difficult to provide direct evidence,so there are still many unanswered questions,such as whether the secondary channel is the only route for NTP to entry and exit,or the main channel could be an alternative.Computational simulation can build a bridge between the macroscopic world and the microscopic world,especially molecular dynamics simulation,which can explain some macroscopic phenomena from the atomic level,such as kinetic.With the development of molecular dynamics simulation,many coarse-grained force fields have emerged,among which the Martini coarse-grained force field is the most popular.Taking RNA polymerase II as an example,thesis explores the conditions and effects of applying Martini coarse-grained molecular dynamics simulations to study protein and substrate binding kinetics.First,the all-atom structure model of RNA polymerase II with a complete transcription bubble was established.After equilibration,it was transformed into a Martini coarse-grained model,and then the binding kinetics of RNA polymerase II and NTP under different elastic network parameters and system concentration settings were studied.Parametric conditions suitable for studying RNA polymerase II-NTP binding kinetics were explored.Analysis of the all-atom structure of the intact RNA polymerase II found that both the secondary channel and the main channel have sufficient space to allow NTP to entry and exit the RNA polymerase II active site,but the secondary channel is more positively electrostatic and more likely attracts negatively charged NTPs into it.Then we randomly added a certain concentration of NTP molecules into the simulation system,and carried out a series of coarse-grained simulations.In the simulation trajectory,we observed the binding-dissociation process of each binding site of NTP in the secondary channel,but the process of NTP molecular diffusion into the main channel was hardly observed,which also indicated that the secondary channel was the main path for NTP diffusion.According to the observation and statistics of the binding kinetics of NTP and RNA polymerase II,we propose a model of NTP entering and exiting the secondary channel that divides the secondary channel into F,H,T,G,E,and A binding sites.It 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.The results of this thesis support that the secondary channel is the only channel for NTP to enter and exit the RNA polymerase II active site,and also demonstrate that Martini coarse-grained simulations can be used as a powerful tool for studying protein-ligand binding kinetics.