Nucleosomal Elements that Control the Topography of the Barrier to Transcription

Bintu, Lacramioara; Ishibashi, Toyotaka; Dangkulwanich, Manchuta; Wu, Yueh-Yi; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos

doi:10.1016/j.cell.2012.10.009

Cited by 163 publications

(225 citation statements)

References 49 publications

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“…We first sought to recapitulate single-molecule results obtained from Pol II transcription experiments along mononucleosomal templates (3,5). We expressed and purified the four canonical yeast histones from Escherichia coli without posttranslational modifications.…”

Section: Resultsmentioning

confidence: 99%

“…Dwell times (the average time the polymerase resides at a base pair) were increased at the nucleosome, in particular within its central part. This part of the nucleosomal DNA corresponds to the dyad region, which is known to be difficult to pass for a polymerase (3,5) (Fig. 1D).…”

Section: Significancementioning

confidence: 99%

mentioning

confidence: 99%

“…In vitro experiments have revealed that a single nucleosome exhibits a significant mechanical barrier to the transcribing Pol II (3)(4)(5)(6)(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).…”

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confidence: 99%

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Nucleosomal arrangement affects single-molecule transcription dynamics

Fitz

Shin

Ehrlich

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Nucleosomal arrangement affects single-molecule transcription dynamics

Fitz

Shin

Ehrlich

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The mechanisms by which RNA polymerases might surpass such roadblocks are poorly understood. Previous single‐molecule studies have focused on RNA polymerases disrupting nucleosomes 1, 2, 3, 4. However, nucleosomes are only found in eukaryotes, interact with DNA nonspecifically, and are substrates for post‐translational modifications that regulate chromatin remodeling and the recruitment of accessory factors that regulate transcription 5.…”

Section: Introductionmentioning

confidence: 99%

Proteins mediating DNA loops effectively block transcription

et al. 2017

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Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop‐mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription.

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Single‐molecule nucleosome remodeling by INO80 and effects of histone tails

et al. 2018

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Genome maintenance and integrity requires continuous alterations of the compaction state of the chromatin structure. Chromatin remodelers, among others the INO80 complex, help organize chromatin by repositioning, reshaping, or evicting nucleosomes. We report on INO80 nucleosome remodeling, assayed by single-molecule Foerster resonance energy transfer on canonical nucleosomes as well as nucleosomes assembled from tailless histones. Nucleosome repositioning by INO80 is a processively catalyzed reaction. During the initiation of remodeling, probed by the INO80 bound state, the nucleosome reveals structurally heterogeneous states for tailless nucleosomes (in contrast to wild-type nucleosomes). We, therefore, propose an altered energy landscape for the INO80-mediated nucleosome sliding reaction in the absence of histone tails.

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Nucleosomal Elements that Control the Topography of the Barrier to Transcription

Cited by 163 publications

References 49 publications

Nucleosomal arrangement affects single-molecule transcription dynamics

Nucleosomal arrangement affects single-molecule transcription dynamics

Proteins mediating DNA loops effectively block transcription

Single‐molecule nucleosome remodeling by INO80 and effects of histone tails

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