RNA Polymerase II (Pol II) is the enzyme responsible for transcribing the genetic information stored in DNA template and synthesizing corresponding messenger RNA for manufacturing proteins in eukaryotes. RNA mutations that escape the proofreading mechanisms of RNA polymerase II may lead to transcriptional mutagenesis and further cause human diseases such as Alzheimer’s disease, Down syndrome, and cancer. RNA Pol II undergoes two critical steps to correct potential mistakes made during transcription: backtracking of the polymerase complex upon mis-incorporation of the wrong nucleotide and the removal of such wrong nucleotide. Recently, we have successfully elucidated dynamic mechanisms of backtracking conformational change upon the misincorporation (Nature Communications, 7, 11244, (2016)). In particular, our simulations coupled with experimental validation demonstrate an important role for the Bridge Helix by serving as a critical checkpoint for the fraying motion through its highly conserved residue T831. This residue can sense the weakened base pairing due to mis-incorporation and further promotes the fraying and subsequent backtracking.
In this paper (Nature Catalysis, (2019), doi: 10.1038/s41929-019-0227-5), we elucidated the reaction mechanism of subsequent cleavage of the mis-incorporated nucleotide to remove dinucleotide at the 3’-terminus. This critical basal endonuclease activity is sufficient to maintain cell viability. Unfortunately, the catalytic mechanism at atomic level of intrinsic cleavage remains unresolved despite extensive efforts. Here, we revealed the catalytic mechanisms of intrinsic cleavage that resolves a long-stranding puzzle: What is the mechanistic origin of the intrinsic proofreading activities by Pol II?
To elucidate the intrinsic cleavage reaction mechanisms of RNA Pol II, we have performed large-scale ab initio QM/MM-MD free energy calculations of a biomolecular system of more than 370,000 atoms. Our work has overcome the limitation of simulations of such large systems that are generally considered to be too computationally expensive to be feasible, demonstrated the power of the state-of-the-art QM/MM-MD approach in solving important biological problems. These calculations are enabled by the Shaheen Supercomputer at KAUST (Ranked #7 worldwide in July 2015). A striking discovery we made is that neither protein residue nor the terminal nucleobase serves as the general base for the catalytic reaction, which contrasts with previous hypotheses. Surprisingly, it is the phosphate oxygen of the terminal nucleotide that plays a crucial role in deprotonating the attacking water molecule. Furthermore, we found that the protonation of the exposed 3’-oxyaion leaving group is assisted by active-site water molecules instead of neighboring protein residues. Our proposed mechanism has been rigorously validated with assay experiments using the RNA template with 2’-5’ linkage modification, as well as nucleobase modification.
Overall, our tightly integrated computational and experimental experiments show that intrinsic cleavage undergoes a generalized mechanism that does not depend on the nucleobase. Our suggested mechanism, in which the phosphate Rp-oxygen serves as the general base rather than any Pol II residues, suggests that Pol II compartmentalizes its backtracking (with conformational change highly reliant on Pol II residues to recognize NTP mis-incorporation) and cleavage mechanisms (Pol II independent reaction mechanism) in order to coordinate these two functions. Our study therefore provides important new insights into understanding how RNA is cleaved by RNA Pol II and structural basis on how it performs proofreading by simultaneous backtracking and intrinsic cleavage.