Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

Abstract

The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop

“…BH GOF variants suppressed strong LOF TL variants Q1078S and H1085Y but failed to suppress, or even exacerbated slightly LOF TL variants H1085Q and F1086S (Fig 6B, 6C and 6E), consistent with conditional epistasis, where GOF activity of BH variants can suppress either specific TL variants or otherwise exert their effects in specific contexts. Finally, recent modeling studies predicted that the BH residue Y836 assists Pol II forward translocation [72] by interacting with the DNA:RNA hybrid. Y836A/H conferred Gal R phenotypes, consistent with LOF and compromised translocation (S10F Fig).…”

Molecular Structures of Transcribing RNA Polymerase I

“…While this region is well resolved in EC1/2, it shows weaker density in the OC and is disordered in the cryo-EM structures of monomeric apo Pol and its initiation-competent form (Engel et al., 2016, Pilsl et al., 2016), suggesting that RNA synthesis, rather than closing of the DNA-binding cleft, triggers the complete folding of the BH. Accordingly, this mobile region includes a conserved threonine A190 T1013 (T831 in Pol II-Rpb1 and T879 in Pol III-C160), which has been proposed as a “probe” for DNA/RNA stability and to be important for TFIIS-stimulated RNA cleavage (Da et al., 2016). In the OC, residue T1013 is in a similar position as in apo Pol I, facing away from base position +1.…”

Kinetics of nucleotide entry into RNA polymerase active site provides mechanism for efficiency and fidelity

“…A more detailed model would consider different concentrations for different NTPs under physiological conditions and differences in kinetic rates and secondary pore interactions as a function of NTP type and require additional simulation data for parametrization. Moreover, we do not consider backtracking and pausing that is believed to occur in a mismatch-dependent manner [10, 45, 52]. The coupling of pyrophosphate release to Pol II dynamics and a possible interference with NTP entry or exit via the secondary pore was also ignored.…”

“…Our model focuses on NTPs but it could be combined with the Pol II-centric kinetic models developed by the Huang group that consider translocation, backtracking and pausing, bridge helix dynamics, and pyrophosphate release [8, 10]. Explicit consideration of translocation, bridge helix dynamics, and pyrophosphate release by itself and possibly coupled with NTP entry would provide a better basis for estimating the A➔C transition rate in contrast to our current model where all of these processes are lumped together with the chemical catalysis step into a single rate that was fit to experimental data.…”

“…High-resolution atomistic structures of RNA polymerases [5] have motivated detailed mechanistic analyses [6] including elaborate computational studies [7–10]. Nevertheless, it has not been fully resolved how exactly RNA polymerase is able to reliably discriminate between cognate and mismatched nucleotides during RNA elongation.…”