Edited by Steven M. Block, Stanford University, Stanford, CA, and approved September 27, 2016 (received for review March 16, 2016)
Transcription is the first step toward protein production. During transcription, a polymerase enzyme moves along DNA and copies it to RNA. In the cell, DNA is highly compacted: roughly 150 bp of DNA is wrapped around histone proteins to form a nucleosome. However, little is known about how closely spaced nucleosomes impact polymerase transcription. We performed single-molecule optical tweezers transcription experiments on dinucleosomal DNA templates. We show that the effect of the second nucleosome on polymerase transcription efficiency through the first one depends on the internucleosomal spacing and the rotational arrangement of the nucleosomes on the helical DNA template. Our findings provide insights into how DNA compaction affects transcription in vitro.
In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.
Author contributions: V.F., P.C., V.Z., and S.W.G. designed research; V.F., J.S., and L.F. performed research; V.F., C.E., and L.F. contributed new reagents/analytic tools; V.F. and J.S. analyzed data; and V.F., J.S., P.C., V.Z., and S.W.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1602764113/-/DCSupplemental.
Freely available online through the PNAS open access option.
