Nucleosomal arrangement affects single-molecule transcription dynamics

Abstract

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.T o accommodate the massive amount of genetic material within the nucleus, DNA is packaged into chromatin. The level of chromatin compaction determines the accessibility of the underlying DNA, which in turn impacts gene expression (1). The first step in gene expression is transcription, where RNA polymerase II (Pol II) processively moves along the DNA template to generate an RNA copy. However, how chromatin impacts the translocation dynamics of individual RNA polymerases has remained unclear.The fundamental unit of chromatin is a single nucleosome, which consists of 147 bp of DNA wrapped ∼ 1.7 times around a histone octamer (2). In vitro experiments have revealed that a single nucleosome exhibits a significant mechanical barrier to the transcribing Pol II (3-7). Using optical tweezers, it was shown that a nucleosome both decreases the rate of forward translocation and increases polymerase pausing and backtracking (3, 5). These changes have been suggested to depend on the unwrapping dynamics of the nucleosome itself, which are governed by nucleosomal properties such as the histones' modification state as well as the underlying DNA sequence (3, 5).In vivo, the process of chromatin transcription is far more complex. Chromatin accessibility is affected by many accessory factors that facilitate the progression of RNA polymerases through nucleosomal obstacles (1). A central outstanding question is to what extent the nucleosomal arrangement affects Pol II transcription behavior. This arrangement might be particularly important in budding yeast, an organism with a compact genome with short internucleosomal distances (8-11).Here, we study the impact of the arrangement of two nucleosomes on the nucleosomal transcription performance of individual molecules of Pol II. We use dual-trap optical tweezers in a single-molecule approach to follow single enzymes of Pol II as they progress through a dinucleosomal array (3, 5). Specifi...

Insight into the unwrapping of the dinucleosome

“…The proposed path takes place along the path I with fluctuations of one or two binding sites. This result is in good agreement with the observation of Fitz et al on the dinucleosome template [34]. They have seen a change in the transcription dynamics of the RNA polymerase II, as well as the variation of the opening forces in the force-extension experiment, in the dinucleosome compared to mono-nucleosome.…”

“…This effect might be seen in the transcription rate of the nucleosomal DNA. It has been observed the transcription rate of RNA polymerase II through DNA becomes faster in the presence of two nucleosomes (dinucleosome case) than for mononucleosome [34].…”

Aging-associated changes in transcriptional elongation influence metazoan longevity

“…4F). These results suggest that reduced precision in the assembly of the chromatin fiber may contribute to changes in Pol-II speed and splicing fidelity (40,44,48) .…”

Optical tweezers studies of transcription by eukaryotic RNA polymerases

“…Assisting force mode is often used in studies of transcription through nucleosomes, where the tension applied to the upstream DNA ensures there is no transfer of nucleosomes behind the polymerase (22)(23)(24).…”

“…They showed that the local nucleosome unwrapping dynamics, and in turn the transcription elongation dynamics, is affected by nucleosomal properties such as the histones modification state, presence of histone tails or the underlying DNA sequence. Recently, our group used optical tweezers to study transcription of Pol II through nucleosomal templates of higher complexity and the influence of nucleosomal geometry on the efficiency of transcription ( Figure 3B) (24). Fitz and colleagues investigated Pol II elongation dynamics along a DNA template with two nucleosomes.…”