Dynamics of Pyrophosphate Ion Release and Its Coupled Trigger Loop Motion from Closed to Open State in RNA Polymerase II

摘要

Pyrophosphate ion (PP_I) release after nucleotidernincorporation is a necessary step for RNA polymerase II (pol Ⅱ) tornenter the next nucleotide addition cycle during transcription elongation.rnHowever, the role of pol II residues in PPj release and the mechanisticrnrelationship between PP_I release and the conformational change of therntrigger loop remain unclear. In this study, we constructed a Markov staternmodel (MSM) from extensive all-atom molecular dynamics (MD)rnsimulations in the explicit solvent to simulate the PP_I release processrnalong the pol II secondary channel. Our results show that the triggerrnloop has significantly larger intrinsic motion after catalysis and formationrnof PP_I, which in turn aids PP_I release mainly through the hydrogenrnbonding between the trigger loop residue H1085 and the (Mg-PP_I)~(2-) group. Once PP_I leaves the active site, it adopts a hoppingrnmodel through several highly conserved positively charged residues such as K752 and K619 to release from the pol II pore regionrnof the secondary channel. These positive hopping sites form favorable interactions with PP_I and generate four kineticallyrnmetastable states as identified by our MSM. Furthermore, our single-mutant simulations suggest that H1085 and K752 aid PP_Irnexit from the active site after catalysis, whereas K619 facilitates its passage through the secondary channel. Finally, we suggest thatrnPP_I release could help the opening motion of the trigger loop, even though PP_I release precedes full opening of the trigger looprndue to faster PP_I dynamics. Our simulations provide predictions to guide future experimental tests.