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

• Main Authors: Manchuta Dangkulwanich, Toyotaka Ishibashi, Shixin Liu, Maria L Kireeva, Lucyna Lubkowska, Mikhail Kashlev, Carlos J Bustamante
• Format: Article
• Language: English
• Published: eLife Sciences Publications Ltd 2013-09-01
• Series: eLife
• Subjects: RNA polymerase II; transcription elongation; Brownian ratchet; translocation; backtracking; optical tweezer
• Online Access: https://elifesciences.org/articles/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.