Impact of Bending and Torsional Deformations of DNA on the Function of T7-RNA Polymerase

Abstract

Transcription of DNA into RNA is the first step in gene expression and is therefore a common target of regulation. The last few decades have witnessed a significant advance in our understanding of the biochemical mechanisms underlying RNA synthesis by RNA polymerase (RNAP). Despite many phenomenological observations establishing a functional relationship between DNA template mechanics and transcription, very little is known about how the mechanical features of DNA interact with RNA polymerase on a mechanistic level. To shed light on how DNA bending and torsional stresses impact RNAP function, we examine the mechanical interaction of bacteriophage T7-RNAP with several circular DNA fragments that differ in length and linking number. In particular, we employ molecular dynamics to simulate the interaction of T7-RNAP with the following three DNA minicircles: 100 base pair(bp) with linking number 9 (Lk=9), 106-bp with Lk=10, and 108-bp with Lk=10. Each of these corresponds respectively to underwound, overwound, and relaxed configurations. The simulations reveal that torque from T7RNAP induces local relaxation of the bend angle of the circular DNA motif. Furthermore, T7RNAP induces out-of-plane deformation of highly strained DNA minicircles. Different torsional stresses seem to impact DNA conformation to various degrees: change in internal torque (undertwisting/overtwisting DNA) forces circular DNA complexed with T7RNAP to contort into a new shape. Based on these observations, we investigate the following: i) how the shape of a supercoiled DNA molecule impacts the barrier for the polymerase translocation step and ii) why DNA twist and bend angles are important factors in determining the transcription rate on strained templates.