Simulation Studies on Nucleotide Selection in T7 RNA Polymerase Transcription

Abstract

Transcription by RNA polymerases (RNAPs) is essential in gene expression. During transcription elongation, an incoming RNA nucleotide binds to the catalytic center of the polymerase, and is added to the end of an existing transcript when the nucleotide correctly pairs with a template DNA nucleotide. Accurate selection of rNTPs is key to maintaining high transcription fidelity. Detailed mechanisms on how the nucleotides are selected are still lack of. using T7 RNAP as a model system, we calculate binding affinities of various NTP molecules with the RNAP elongation complex, in both its insertion and pre-insertion states. Stable complex structures are obtained by using molecular dynamics simulation. Techniques such as MM-GB/SA, QM/MM-GB/SA are applied to compare the rNTP binding affinities. In addition, free energy perturbation and thermodynamic integration methods are also utilized to calculate the rNTP binding free energies. Based on the simulation results, we would be able to decide which checkpoints (pre-insertion and/or insertion state) the RNAP utilizes essentially to select against wrong nucleotides. We would also like to know how each selection proceeds (rejection and/or inhibition), as well as how strong each selection is in terms of the binding free energy differences between right and wrong nucleotides.