Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism
Manchuta Dangkulwanich,
Toyotaka Ishibashi,
Shixin Liu,
Maria L Kireeva,
Lucyna Lubkowska,
Mikhail Kashlev,
Carlos J Bustamante
Affiliations
Manchuta Dangkulwanich
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
Toyotaka Ishibashi
Jason L Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, United States; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States
Shixin Liu
Jason L Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, United States; Department of Physics, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
Maria L Kireeva
Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research–National Cancer Institute, Frederick, United States
Lucyna Lubkowska
Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research–National Cancer Institute, Frederick, United States
Mikhail Kashlev
Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research–National Cancer Institute, Frederick, United States
Carlos J Bustamante
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; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States; Department of Physics, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States
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.
