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Open Access Publications from the University of California

Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism.

  • Author(s): Dangkulwanich, Manchuta;
  • Ishibashi, Toyotaka;
  • Liu, Shixin;
  • Kireeva, Maria;
  • Lubkowska, Lucyna;
  • Kashlev, Mikhail;
  • BUSTAMANTE, Carlos J.
  • et al.

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:

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