PPi Release Followed by DNA Translocation Studied from Atomistic Simulations of T7 RNA Polymerase Transcription

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. We computationally study the pyrophosphate ion (PPi) product release and DNA translocation processes during the T7 RNAP elongation with unprecedented structural dynamics detail. We implemented extensive all-atom molecular dynamics (MD) simulations and constructed the Markov state model (MSM). The MSM reveals a jump-from-cavity PPi release mechanism, which distinguishes from charge facilitated hopping mechanisms identified previously for bacterial and eukaryotic multi-subunit RNAPs. Furthermore, we performed steered MD simulations and microsecond long MD simulations to explore additional couplings and slow motions. We found that the PPi release does not appear to be tightly coupled to opening of an O-helix that is directly tied to the DNA translocation. Hence, the study disfavors a power stroke mechanism of the RNAP elongation. In particular, we discovered a key lysine/arginine residue that assists the PPi release and the module appears to be universal not only to structurally similar polymerases but also to the multi-subunit RNAPs, even though their overall structures and mechanisms look quite different. We also want to understand why the viral RNAP does not backtrack during its Brownian ratcheting.