Translocation And Selection Mechanisms Of RNA Polymerases By Molecular Dynamics Simulations

RNA polymerase(RNAP)plays a critical role in the expression of genetic information: transcribing DNA to RNA by translocating along the DNA strands.RNAP contains two types: single-subunit versions and multi-subunit versions.During the transcription process,the two most important processes are translocation process and substrate selection process.RNAP can transcribe hundreds to thousands of nucleotides at one time,and the mechanical translocation mechanism of RNAP has been a hotspot and focus in this ongoing process.The fidelity of genetic information is critical to the living organism,which is related to fidelity during the transcription process is the selection mechanism of RNAP for the substrate nucleotides.In recent decades,researches have provided a lot of information about the working mechanism of RNAP,but there are still many places that are unclear.Molecular dynamics(MD)simulation plays an irreplaceable role in recent years as an important supplement to the experiment.In this paper,the method of molecular dynamics simulation is used to study the translocation and selection mechanisms of RNA polymerase,which provides important information for the study of its mechanisms.The full text mainly includes the following parts:Study on translocation mechanism of Pol II.Comparisons of the structure of RNA polymerase II(Pol II)with that of bacterial enzyme have suggested that the transition of the bridge helix(BH)from straight to flipped-out conformations facilitates the translocation of upstream DNA-RNA hybrid.However,the flipped-out conformation of BH in Pol II has not been observed up to now.Using MD simulations,we found two BH flipped-out structures,both of which could facilitate the upstream hybrid translocation.One of the structures shows nearly identical to the crystal flipped-out BH conformation in bacteria,which demonstrates the presence of the flipped-out BH conformation in Pol II.Study on the translocation mechanism of T7 RNAP.Phage T7 RNAP is a prototype single-subunit RNAP.There are mainly two hypothesis related to its translocation mechanism: Power-Stroke model and Brownian ratchet model.The results of our MD simulation showed that the translocation mechanism of T7 RNAP is via a Brownian Ratchet model.We further explained the source of the energy difference before and after the translocation(pre-translocation and post-translocation states)in the experiment.The calculated results are in good agreement with the experimental results.Study on the selection mechanism of T7 RNAP.We found that there was an intermediate state of the enzyme conformation during the T7 RNAP translocation process by MD simulations.In this intermediate state,the transition DNA nucleotide(TN)to be paired as template can be shifted to the active site and participate in the selection of the substrate NTP.This structure does not have a corresponding crystal structure.NTP selection in this intermediate state was studied,revealing that the selection in the intermediate state can be achieved relying on the effect of WatsonCrick interaction between NTP and template DNA nucleotide,effect of stability of the components near the active site and role of tyrosine 639.This indicates that another NTP-selection pathway can also exist besides the main pathway where NTP selection begins at the post-translocation state upon the entry of NTP.