A method for quantifying the force dependence of initiation by T7 RNA polymerase

Abstract

To access the genetic code to be transcribed to RNA, RNA polymerases must first open a "transcription bubble" in the DNA. Structural studies suggest that the minimal model of initiation by T7 bacterophage RNA polymerase (T7 RNAP) consists of two distinct steps: initial binding, in which the T7 RNAP binds to and bends the DNA, and opening, achieved by "scrunching" of the DNA. Since both steps involve mechanical deformation of the DNA, both may be affected by downstream DNA tension. Using an oscillating two-bead optical tweezers assay, we have measured the lifetime of single T7 RNAP-DNA initation complexes under tension. Global maximumlikelihood fitting of force-dependent and non-force-dependent versions of this minimal model shows that there is no conclusively discernible force-dependence of initiation in the measured 0-2 pN DNA tension range.

Downstream DNA Tension Regulates the Stability of the T7 RNA Polymerase Initiation Complex

“…To further determine the nature of the tension dependence, we evaluated the relative fitness of the various possible kinetic models using information theory (26). We estimated the rates and distances to the transition state by fitting the wait-time probability distribution function, w(t), shown below, to the data by numerically maximizing the likelihood using the ga() function of MATLAB (The MathWorks, Natick, MA) with the constraint that the rates be positive:…”