A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control

Abstract

RNA polymerase (RNAP) from bacteriophage T7 is a representative single-subunit viral RNAP that can transcribe with high promoter activities without assistances from transcription factors. We accordingly studied this small transcription machine computationally as a model system to understand underlying mechanisms of mechano-chemical coupling and fidelity control in the RNAP transcription elongation. Here we summarize our computational work from several recent publications to demonstrate first how T7 RNAP translocates via Brownian alike motions along DNA right after the catalytic product release. Then we show how the backward translocation motions are prevented at post-translocation upon successful nucleotide incorporation, which is also subject to stepwise nucleotide selection and acts as a pawl for “selective ratcheting”. The structural dynamics and energetics features revealed from our atomistic molecular dynamics (MD) simulations and related analyses on the single-subunit T7 RNAP thus provided detailed and quantitative characterizations on the Brownian-ratchet working scenario of a prototypical transcription machine with sophisticated nucleotide selectivity for fidelity control. The presented mechanisms can be more or less general for structurally similar viral or mitochondrial RNAPs and some of DNA polymerases, or even for the RNAP engine of the more complicated transcription machinery in higher organisms.

Markov State Models and Molecular Dynamics Simulations Provide Understanding of the Nucleotide-Dependent Dimerization-Based Activation of LRRK2 ROC Domain

“…Similar structural transitions were also observed in motifs mediating Gnt2 binding. Such changes in regions implicated in nucleotide binding toward a “closed” conformation during the exchange of GDP with GTP in the activation cycle were also observed for classical GTPase including Ras [ 38 ], and their similarities could provide guidance on further investigations regarding the detailed GTP hydrolysis process by the ROC homodimer of LRRK2.…”

“…To obtain a comprehensive insight into the dynamics of ROC homodimer during nucleotide shift, we employed large-scale all-atom molecular dynamics (MD) simulation to observe in silico at the atomistic level the intrinsic regulation of GTPase activation cycle of the ROC domain of LRRK2. Furthermore, by integrating our extensive MD simulations with the statistically robust Markov state models (MSMs) for interpreting configuration sampling [ 38 , 39 , 40 ], kinetically relevant states of ROCs homodimer as well as their interconversion rates during GDP/GTP exchange are accessible. Based on MSMs, our study provided for the first time the dynamic portrait of ROCs during nucleotide turnover, as characterized by the previously unreported coupling between dimerization and nucleotide-binding state, with the ROCs dimer exhibiting the “open” conformation with greater dimerization extent in the presence of GDP but switching to the “closed” structure with oligomerization tendency while in complex with GTP.…”

Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

“…It should be pointed out that the triphosphate reorientation of the incoming nucleotide had been suggested for the PV RdRp fidelity control [60]. Additionally, it is interesting to notice that motif F-R555 structurally corresponds to R174 from PV RdRp and R158 in HCV RdRp [15], as well as to Y639 from T7 RNAP that is key to nucleotide selectivity and polymerase translocation [35]. Overall, the ATP insertion seems to be facilitated by an insertion path with quenched fluctuations on the +1 template nt for stabilized base pairing, while the RDV-TP insertion dominated by the template base-stacking populations, is supported by freely fluctuating template nt, leading to transition to the highly stabilized base pairing configurations, with an insertion free energy barrier as low as ∼1.5 kcal/mol or ∼2-3k B T, marginally above thermal fluctuations.…”

“…In several such polymerase species, the nucleotide insertion is rate-limiting (or partially rate-limiting) [33,70,71], thus being critical for nucleotide selection [34]. Comparing to phage T7 RNAP we studied previously [35,36] Current simulations comparing RDV-TP and ATP thus suggest that the nucleotide insertion is coordinated by +1 template nt as well as some interactions on the nucleotide upon initial binding, in which the triphosphate stabilization and re-positioning appear to be essential.…”

“…milliseconds or above), i.e., to be rate limiting (or partially rate-limiting) in the NAC [31][32][33]. Such a slow nucleotide insertion step correspondingly plays a significant role in the nucleotide selection or fidelity control, for example, in the single-subunit viral T7 RNAP system studied recently [34][35][36].…”