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.
Published Web Locationhttps://doi.org/10.7554/eLife.00971
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.