Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate

Abstract

RNA polymerase II (Pol II) utilises the same active site for polymerization and intrinsic cleavage. Pol II proofreads the nascent transcript by its intrinsic nuclease activity to maintain high transcriptional fidelity critical for cell growth and viability. The detailed catalytic mechanism of intrinsic cleavage remains unknown. Here, we combined ab initio quantum mechanics/molecular mechanics studies and biochemical cleavage assays to show that Pol II utilises downstream phosphate oxygen to activate the attacking nucleophile in hydrolysis, while the newly formed 3’-end is protonated through active-site water without a defined general acid. Experimentally, alteration of downstream phosphate oxygen either by 2’−5’ sugar linkage or stereo-specific thio-substitution of phosphate oxygen drastically reduced cleavage rate. We showed by N7-modification that guanine nucleobase does not directly involve as acid-base catalyst. Our proposed mechanism provides important insights into the understanding of intrinsic transcriptional cleavage reaction, an essential step of transcriptional fidelity control.

1′-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition and proofreading during SARS-CoV-2 viral RNA replication

“…To assess the stability of cleavage site, we examined the distance between MgA and the O3' atom of the second last nucleotide at the 3'-terminal as it is essential for cleavage (67-69) (Figure 5A). This distance is well maintained in majority of the simulations for the wildtype-RNA (Supplementary Figure S11), and the histogram shows the highest peak in the region of 2.0~2.5 Å (Figure 5A) suitable for cleavage (67)(68)(69). By sharp contrast, MD simulations with Remdesivir at 3'-terminal show destabilization of cleavage site in ExoN, with the increased height of the second peak at ~3.7 Å (Figure 5A and Supplementary Figure S12).…”

Catalytic Mechanism of Non-Target DNA Cleavage in CRISPR-Cas9 Revealed by Ab Initio Molecular Dynamics

“…The choice of simulating an enlarged QM partincluding six solvation waters and protein residues, as well as the adjacent DNA phosphate groupsenabled to assess whether these chemical groups engage in the catalysis and the related proton transfer events. This is a key point because the activation of the nucleophile could occur in different ways, such as through an amino acid , or a phosphate group − serving as a general base or even through the bulk water. , Notably, these ab initio simulations have been performed by substituting the crystallized Mn 2+ ions with the catalytically active Mg 2+ ions …”

RNA polymerase II stalls on oxidative DNA damage via a torsion-latch mechanism involving lone pair–π and CH–π interactions

“…From the bottom of the Gh lesion, we found the hydrogen bonding between the C4 carbonyl group of the Gh hydantoin ring and conserved Thr831 (Rpb1) from the bridge helix motif further fix the hydantoin ring of Gh lesion. Thr831 is a strictly conserved residue in the bridge helix and has an important role for Pol II translocation (45)(46)(47).…”