Significance: In this study, by using molecular dynamics simulations and Markov state models, we reveal that RNA polymerase II translocation is driven purely by thermal energy and does not require the input of any additional chemical energy. Our simulations show an important role for the bridge helix: Large thermal oscillations of this structural element facilitate the translocation by specific interactions that lower the free-energy barriers between four metastable states. Among these states, we identify two previously unidentified intermediates that have not been previously captured by crystallography. The dynamic view of translocation presented in our study represents a substantial advance over the current understanding based on the static snapshots provided by X-ray structures of transcribing complexes.

The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

The dynamics of the PP_i release during the transcription elongation of bacterial RNA polymerase and its effects on the Trigger Loop (TL) opening motion are still elusive. Here, we built a Markov State Model (MSM) from extensive all-atom molecular dynamics (MD) simulations to investigate the mechanism of the PP_i release. Our MSM has identified a simple two-state mechanism for the PP_i release instead of a more complex four-state mechanism observed in RNA polymerase II (Pol II). We observed that the PP_i release in bacterial RNA polymerase occurs at sub-microsecond timescale, which is ∼3-fold faster than that in Pol II. After escaping from the active site, the (Mg-PP_i)²⁻ group passes through a single elongated metastable region where several positively charged residues on the secondary channel provide favorable interactions. Surprisingly, we found that the PP_i release is not coupled with the TL unfolding but correlates tightly with the side-chain rotation of the TL residue R1239. Our work sheds light on the dynamics underlying the transcription elongation of the bacterial RNA polymerase. Pyrophosphate ion (PP_i) release is a critical step in the nucleotide addition cycle of transcription elongation. Despite extensive experimental studies, the kinetic mechanism of the PP_i release in bacterial RNA polymerases (RNAP) still remains largely a mystery. As a cellular machine, RNAP contains more than 3000 residues, and thus it is challenging for all-atom molecular dynamics (MD) simulations to directly capture the process of the PP_i release. In this study, we have simulated the dynamics of the PP_i release at microsecond timescale using the Markov State Models (MSMs) built from extensive MD simulations in explicit solvent. MSM is a powerful kinetic network model and can predict long timescale dynamics from many short MD simulations. Our results suggest a simple two-state model for the PP_i release in RNAP, which sharply contrasts with the more complex four-state hopping model in the yeast RNA polymerase (Pol II). We also observe a 3-fold faster dynamics for the PP_i release in RNAP compared to Pol II, consistent with the faster transcription rate in the bacterial systems. Our results greatly improve our understanding of the PP_i release, and also provide predictions to guide future experimental tests.