A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation

Pyrophosphate ion (PPi) release during transcription elongation is a signature step 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 in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. 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 of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase.Author Summary: RNA polymerases (RNAPs) are usually recognized to work as Brownian ratchet machines, with mechanical movements and chemical reactions loosely coupled during transcription elongation. Nevertheless, structural studies on the single-subunit T7 RNAP had suggested an alternative power stroke scenario, which requires the PPi product release reaction tightly couples with or drives the polymerase translocation. To resolve the conflicting views in the mechano-chemical coupling scenario, we conducted atomistic molecular dynamics (MD) simulations to investigate the PPi release mechanism of T7 RNAP. Using a large number of nanosecond short simulations, we constructed the Markov state model (MSM) and found that the PPi release undergoes a jump-from-cavity activation process, unlikely to further support the translocation. The MSM implementations to multi-subunit RNAPs previously demonstrated instead the charge facilitated hopping mechanisms of the PPi release. To further explore essential slow motions during the PPi release, we also performed microsecond long MD simulations. The PPi release does not appear to be tightly coupled to the rotational opening of an O-helix that is directly tied to the polymerase translocation. Hence, the study again disfavors the power stroke scenario. Remarkably, we discovered a key residue Lys472 that is able to assist PPi to jump out of the cavity. The mechanism can be general to a group of polymerases sharing conserved structural features with T7 RNAP, while the lysine or arginine module to assist the PPi release can be universal to both the single and multi-subunit RNAPs, even though the overall structural features and the PPi release mechanisms are significantly different.