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Title: 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.
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [4] ;  [4] ;  [5]
  1. 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
  2. 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
  3. 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
  4. Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research–National Cancer Institute, Frederick, United States
  5. 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
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Published Article
Journal Name:
eLife
Additional Journal Information:
Journal Name: eLife Journal Volume: 2; Journal ID: ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
OSTI Identifier:
1197625
Alternate Identifier(s):
OSTI ID: 1197626

Dangkulwanich, Manchuta, Ishibashi, Toyotaka, Liu, Shixin, Kireeva, Maria L., Lubkowska, Lucyna, Kashlev, Mikhail, and Bustamante, Carlos J.. Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. United States: N. p., Web. doi:10.7554/eLife.00971.
Dangkulwanich, Manchuta, Ishibashi, Toyotaka, Liu, Shixin, Kireeva, Maria L., Lubkowska, Lucyna, Kashlev, Mikhail, & Bustamante, Carlos J.. Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. United States. doi:10.7554/eLife.00971.
Dangkulwanich, Manchuta, Ishibashi, Toyotaka, Liu, Shixin, Kireeva, Maria L., Lubkowska, Lucyna, Kashlev, Mikhail, and Bustamante, Carlos J.. 2013. "Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism". United States. doi:10.7554/eLife.00971.
@article{osti_1197625,
title = {Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism},
author = {Dangkulwanich, Manchuta and Ishibashi, Toyotaka and Liu, Shixin and Kireeva, Maria L. and Lubkowska, Lucyna and Kashlev, Mikhail and Bustamante, Carlos J.},
abstractNote = {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 = {10.7554/eLife.00971},
journal = {eLife},
number = ,
volume = 2,
place = {United States},
year = {2013},
month = {9}
}