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

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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 Liu1, Manchuta Dangkulwanich 1, Toyotaka Ishibashi1, Maria Kireeva2, Lucyna
Lubkowska2 , Mikhail Kashlev2, Carlos Bustamante1,3.
1University of California, Berkeley, Berkeley, CA, USA, 2National Cancer Institute, Frederick,
MD, USA, 3Howard 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.