The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II has been investigated using molecular mechanics and quantum mechanics methods.Molecular dynamics(MD) simulations were carried out using the TIP3 water model and generalized solvent boundary potential(GSBP) by CHARMM based on the X-ray crystal structure.Two models of the ternary elongation complex were constructed based on CHARMM MD calculations.All the species including reactants,transition states,intermediates,and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2.Three pathways were explored using the DFT method.The most favorable reaction pathway involves indirect proton migration from the RNA primer 3’-OH to the oxygen atom of-phosphate via a solvent water molecule,proton rotation from the oxygen atom of-phosphate to the-phosphate side,the RNA primer 3’-O nucleophilic attack on the-phosphorus atom,and P-O bond breakage.The corresponding reaction potential profile was obtained.The rate limiting step,with a barrier height of 21.5 kcal/mol,is the RNA primer 3’-O nucleophilic attack,rather than the commonly considered proton transfer process.A high-resolution crystal structure including crystallographic water molecules is required for further studies. The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II has been investigated using molecular mechanics and quantum mechanics methods. Molecular dynamics (MD) simulations were carried out using the TIP3 water model and generalized solvent boundary potential (GSBP) by CHARMM based on the X-ray crystal structure. Two models of the ternary elongation complex constructed on CHARMM MD calculations. All the species including reactants, transition states, intermediates, and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2. Pathways were explored using the DFT method. The most favorable reaction pathway contains indirect proton migration from the RNA primer 3’-OH to the oxygen atom of-phosphate via a solvent water molecule, proton rotation from the oxygen atom of-phosphate to the-phosphate side, the RNA primer 3’-O nucleophilic attack on the-phosphorus atom, and PO bond breakage. The correspo nding reaction potential profile was obtained. The rate limiting step, with a barrier height of 21.5 kcal / mol, is the RNA primer 3’-O nucleophilic attack, rather than the commonly considered proton transfer process. A high-resolution crystal structure including crystallographic water molecules is required for further studies