The Catalytic Mechanism of RNA Polymerase II

Eukaryotic RNA polymerase II (RNAP II) transcribes the DNA into mRNA. The presence of two metal ions (usually Mg2+) and conserved aspartate residues in the active sites of all nucleic acid polymerases led to the adoption of a universal catalytic mechanism, known as the two metal ion catalysis. In this scheme, it is assumed that the coordination shell of Mg2+ (geometry, number, and identity of the ligands) is basically the same for all of the enzymes, despite the significant differences in sequence and structure commonly found in multisubunit RNA polymerases versus single-subunit RNA polymerases and DNA polyinerases. Here, we have studied the catalytic mechanism of RNAP II and found very interesting variations to the postulated mechanism. We have used an array of techniques that included thermodynamic integration free energy calculations and electronic structure calculations with pure DFT as well as hybrid DFT/semiempirical methods to understand this important mechanism. We have studied four different catalytic pathways in total, resulting from different combinations of proton donors/acceptors for the two proton transfers experimentally detected (deprotonation of the 3' hydroxyl of the terminal nucleotide (HORNA) and protonation of pyrophosphate). The obtained data unambiguously show that the catalytic mechanism involves the deprotonation of HORNA by a hydroxide ion corning from the bulk solvent, the protonation of pyrophosphate by the active site His1085, and the nucleophilic attack to the substrate by O-RNA(-). The overall barrier is 9.9 kcal/mol. This mechanism differs from those proposed in the identity of the general acid. The deprotonation of the HORNA and the transition state for the nucleophilic attack are similar to some (but not all) of the family members.