In eukaryotic cells, RNA polymerase II (Pol II) transcribes the information encoded in DNA into messenger RNA. In the cell, external and internal factors constantly damage DNA. If the damage is not detected, it can lead to mis-incorporations during transcription. Nevertheless, Pol II can detect and cleave mis-incorporations in a proofreading mechanism that is one of the critical components to maintain the high fidelity of transcription. When there is only one mismatch, the misincorporated RNA nucleotide would be removed as a dinucleotide without involving other transcription factors. In this work, quantum mechanics/molecular mechanics (QM/MM) calculations with reaction coordinate driving method were used to identify a possible mechanism for the intrinsic cleavage in backtracked RNA poly...[ Read more ]

In eukaryotic cells, RNA polymerase II (Pol II) transcribes the information encoded in DNA into messenger RNA. In the cell, external and internal factors constantly damage DNA. If the damage is not detected, it can lead to mis-incorporations during transcription. Nevertheless, Pol II can detect and cleave mis-incorporations in a proofreading mechanism that is one of the critical components to maintain the high fidelity of transcription. When there is only one mismatch, the misincorporated RNA nucleotide would be removed as a dinucleotide without involving other transcription factors. In this work, quantum mechanics/molecular mechanics (QM/MM) calculations with reaction coordinate driving method were used to identify a possible mechanism for the intrinsic cleavage in backtracked RNA polymerase II (Pol II). Our results suggest that the phosphate group of the mismatch nucleotide deprotonates a nearby metal-coordinating water molecule to generate the attacking nucleophile. S_N2 cleavage of the penultimate phosphodiester bond results in a metastable intermediate state. The exposed 3’-end oxygen is then protonated by proton transfer through two other coordinating water molecules, leading to product formation. This mechanism also explains why endonuclease is predominant over exonuclease activity in Pol II.