Abstract
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.
Highlights
Transcription elongation is a continuous process of producing messenger RNA as an RNA polymerase (RNAP) moves along double stranded DNA, copying information from the DNA template to synthesize an RNA strand [1,2,3]
From a hundred of short molecular dynamics (MD) simulations, we built a kinetic model of the pyrophosphate ion (PPi) release by constructing the Markov state model (MSM)
In this work we studied the process in structural dynamics detail by implementing atomistic MD simulations based on the high-resolution structure of T7 RNAP product complex
Summary
Transcription elongation is a continuous process of producing messenger RNA as an RNA polymerase (RNAP) moves along double stranded (ds) DNA, copying information from the DNA template to synthesize an RNA strand [1,2,3]. In studying the RNAP structures from bacteriophage T7, it was suggested that the PPi release directly drives the polymerase translocation [7]. Such a mechanism is called the ‘power stroke’ (PS), as the product release reaction couples tightly to the mechanical movement of the protein [10,11]. Single molecule measurements implementing optical-tweezer forces on RNAPs, for single or multi-subunit ones, consistently suggested a ‘Brownian ratchet’ (BR) mechanism [12,13,14], in which the translocation proceeds in Brownian motions, thermally activated without being energetically coupled to the preceding PPi release. Studies that are able to zoom into structural detail and to characterize kinetic or energetics of the process are highly demanded for understanding the basics of the transcription engine
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