Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

27-POS Board 27 Complete Kinetic Dissection Reveals the Rate-Limiting Mechanism of Transcription Elongation by RNA Polymerase II Shixin Liu 1 , Manchuta Dangkulwanich 1 , Toyotaka Ishibashi 1 , Maria Kireeva 2 , Lucyna Lubkowska 2 , Mikhail Kashlev 2 , Carlos Bustamante 1,3 . 1 University of California, Berkeley, Berkeley, CA, USA, 2 National Cancer Institute, Frederick, MD, USA, 3 Howard Hughes Medical Institute, Berkeley, CA, USA. RNA polymerase is a molecular motor that converts chemical energy from ribonucleotide incorporation into mechanical translocation along the DNA template. It is generally accepted that such mechanochemical coupling occurs by a "Brownian ratchet" instead of a "power stroke" mechanism. However, the rate-limiting steps within the Brownian ratchet framework remained controversial. By using single-molecule manipulation and challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the subsequent catalysis rate, in contradiction to the prevalent assumption that the pre- and post- translocated states rapidly reach equilibrium before catalysis. This finding reveals a linear Brownian ratchet mechanism in which translocation represents one of the rate-limiting steps. This mechanism unifies structural, biochemical, and single-molecule data and suggests that the recently proposed branched ratchet model, which necessitates a putative secondary nucleotide binding site on the enzyme, is not required to explain the observed force-velocity dependence of the polymerase. We further determined the other major on- and off-pathway kinetic parameters in the elongation cycle. The resulting energy landscape shows that the off-pathway states are favored thermodynamically, but not kinetically, over the on-pathway states, conferring the polymerase its propensity to pause and providing a physical basis for transcriptional regulation.

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