Ratcheting Mechanism of T7 RNA Polymerase During Transcription Elongation

Abstract

RNA polymerases (RNAPs) can act as molecular motors, moving processively along DNA and synthesizing RNA from the DNA template. The RNAP from bacteriophage T7 is a single subunit enzyme that can carry out all transcriptional functions in the absence of additional protein factors, from initiation, elongation to termination. The structure of T7 RNAP resembles some of DNA polymerases in a hand-like configuration. In this modeling study we focus on how T7 RNAP couples its translocation with nucleotide addition during transcription elongation. Based on structural information and experimental data from single molecule force measurements, we build a ‘facilitated’ Brownian ratchet model that may resolve a debate between ‘power stroke’ and ‘Brownian ratchet’ views of this RNAP. In present model, the RNAP translocation is achieved either by thermal hopping, or by a facilitated movement upon Tyr639 side chain ‘pushing’. Under this scenario, we examine as well how the RNAP efficiently selects right nucleotide starting from the nucleotide pre-insertion, a ‘ratchet’ step right after the translocation. Both O-helix and Tyr639 seem to play important roles in the nucleotide selection. The selection accounts fully for transcription fidelity of T7 RNAP due to lack of proof reading. Further, we incorporate sequence effect into the translocation model and probe intrinsic termination of T7 RNAP through a forward tracking intermediate. The termination efficiency seems to be boosted by the facilitated translocation. Our present scenario for T7 RNAP is similar to a ‘two-paw’ ratchet mechanism proposed for multi-subunit RNAP. Accordingly, functional analogies could be made between involved structural elements from the two types of RNAPs.