Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism.
During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By 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 catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation. DOI:http://dx.doi.org/10.7554/eLife.00971.001.
Jason L Choy Laboratory of Single-Molecule Biophysics , University of California, Berkeley , Berkeley , United States ; Department of Chemistry , University of California, Berkeley , Berkeley , United States.
Manchuta
M
Dangkulwanich
Toyotaka
T
Ishibashi
Shixin
S
Liu
Maria L
ML
Kireeva
Lucyna
L
Lubkowska
Mikhail
M
Kashlev
Carlos J
CJ
Bustamante
D007700:Kinetics; D008962:Models, Theoretical; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic
D012319:RNA Polymerase II
Brownian ratchet; RNA polymerase II; S. cerevisiae; backtracking; optical tweezers; transcription elongation; translocation