20 August 2009 A method for quantifying the force dependence of initiation by T7 RNA polymerase
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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.
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Bennett S. Kalafut, Gary M. Skinner, Koen Visscher, "A method for quantifying the force dependence of initiation by T7 RNA polymerase", Proc. SPIE 7400, Optical Trapping and Optical Micromanipulation VI, 740005 (20 August 2009); doi: 10.1117/12.827659; https://doi.org/10.1117/12.827659
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