The Mechanism Of Pyrophosphate Ion Release And Polymerase Translocation From Atomistic Simulations Of Single-subunit RNA Polymerase Transcription

Transcription and replication are most fundamental and essential processes in molecular genetics and evolution,conducted by molecular motors in charge of the processes,i.e.,mainly RNA/DNA polymerase,that work at non-equilibrium steady state.Bacteriophage T7 RNA polymerase(RNAP)serves a prototypical system to study general physical mechanisms of transcription due to its relative simple structures along with self-sufficient and strong transcription activities.Pyrophosphate ion(PPi)release and polymerase translocation during transcription elongation are the signature and key steps in each nucleotide addition cycle.The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation.Here we investigated detailed dynamics of the PPi release process and the RNAP translocation in the single-subunit T7 RNAP,implementing all-atom molecular dynamics(MD)simulations to build the Markov state model(MSM).We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations.We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids,followed by a comparatively slow jump-from-cavity activation process.Combining with a number of microsecond long MD simulations,we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions.On the other hand,the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out.The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues.Remarkably,the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms.We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain along with the translocation of T7 RNAP.Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of T7 RNAP.Currently,we study the translocation mechanism of T7 RNAP similarly,using extensive all-atom MD simulations and constructed the MSM.The MSM construction reveals a translocation pathway with the O-helix opening indispensable for the translocation.In particular,we have found that the opening of the O-helix facilitates the upstream RNA-DNA translocation by pushing the synthesizing RNA 3'-end.We also study why this T7 RNAP does not backtrack during its transcription elongation as in other RNAPs from bacteria and eukaryotes.We suggest it is,the O-helix opening in the pre-translocation state that inhibits the RNAP back-tracking.We are designing mutant T7 RNAPs in collaboration with experimental approatches to prove the suggestd mechanishs.