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

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.

The hepatitis C virus RNA-dependent RNA polymerase directs incoming nucleotides to its active site through magnesium-dependent dynamics within its F motif

“…1 and 2 in Fig. 8 are distinct states) (29). This finding is consistent with the emerging picture that catalysis and translocation are actually uncoupled in base polymerases including RdRp (30).…”

A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control

“…We have recently studied transcription elongation of T7 RNAP by combining physical modeling and all-atom MD simulations, addressing both mechano-chemical coupling and fidelity control mechanisms during elongation [ [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] ]. The mechanochemistry concerns about how the protein machine utilizes chemical free energy to generate mechanical or directional motions, referring to how the chemical synthesis of RNA couples with the polymerase translocation along DNA in the RNAP system.…”

“…On the other hand, early [ 39 ] and immediately later single-molecule force measurements on T7 RNAP suggested alternatively the BR scenario [ 25 ]. Accordingly, we investigated the mechano-chemical coupling by studying the PPi release first, using atomistic MD simulation [ 31 ].…”

“…For systems of such a size, one can routinely simulate up to several microseconds under current high-performance computing technologies; yet it is still computationally prohibiting to further approach over tens of microseconds to millisecond time scale. Fortunately, by launching extensive sub-microseconds equilibrium simulations that spread around a wide range of conformation space for the relevant process, we were able to construct the Markov-state model (MSM) for the PPi release, and later for the translocation of T7 RNAP, which are estimated to happen at tens of microsecond time scale [ 31 , 34 ]. The strength and technical issues in building the MSM using MD can be found in abundant literature elsewhere [ [46] , [47] , [48] , [49] , [50] ].…”

RNA Polymerase interactions and elongation rate

“…The major and generic features of the multi-step mechano-chemical pathway of individual RNAP motors during each elongation step are: (1) Nucleoside triphosphate (NTP) binding at a catalytic site within the transcription bubble, (2) NTP hydrolysis, (3) Release of pyrophosphate (PP i , one of the products of hydrolysis), (4) Forward step of the RNAP along the DNA template by one base pair (bp) [2,6]. Recently, the mechanism of NTP binding to the active site [7] and PP i release [8] were studied with atomistic simulations in great detail for T7 RNA Polymerase. Also recently, the role of trigger loop folding-unfolding on the RNAP translocation was elucidated for the RNAP of Escherichia coli [9].…”