Burgos-Bravo Francesca, Tong Alexander B, Li Chen, Díaz-Celis César, Kaplan Craig D, LeRoy Gary, Reinberg Danny, Bustamante Carlos
Jason Choy Laboratory of Single Molecule Biophysics, University of California, Berkeley, Berkeley, CA 94704, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94704, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
Jason Choy Laboratory of Single Molecule Biophysics, University of California, Berkeley, Berkeley, CA 94704, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
Mol Cell. 2025 Jun 5;85(11):2097-2109.e8. doi: 10.1016/j.molcel.2025.05.002. Epub 2025 May 23.
Transcription of yeast RNA polymerase II through nucleosomes requires the assistance of the histone chaperone FACT (facilitates chromatin transcription). Yet, how FACT modulates the nucleosomal mechanical barrier to affect the polymerase's elongation dynamics is poorly understood. Using high-resolution single-molecule optical tweezers, we show that FACT greatly decreases the magnitude of the barrier by favoring the unwrapping of DNA from the distal H2A-H2B dimer, which, in turn, weakens the contacts near the dyad, significantly reducing the enzyme's crossing time. We show that barrier crossing depends on the asymmetric flexibility of the nucleosome arms, an asymmetry we find across the genome. Mechanical unwrapping of Cy3-H2A nucleosomes reveals that FACT reduces their unwrapping force and stabilizes a hexasome-like intermediate that retains both labeled dimers during successive unwrapping cycles. This intermediate is also observed after transcription. In conclusion, FACT facilitates nucleosomal transcription by weakening the barrier and actively assisting the maintenance of nucleosomal integrity after enzyme passage.
酵母RNA聚合酶II转录通过核小体需要组蛋白伴侣FACT（促进染色质转录）的协助。然而，FACT如何调节核小体的机械屏障以影响聚合酶的延伸动力学仍知之甚少。使用高分辨率单分子光镊，我们发现FACT通过促进DNA从远端H2A-H2B二聚体上解旋，极大地降低了屏障的大小，这反过来又削弱了二分体附近的接触，显著减少了酶的穿越时间。我们表明，穿越屏障取决于核小体臂的不对称柔韧性，我们在全基因组中都发现了这种不对称性。Cy3-H2A核小体的机械解旋表明，FACT降低了它们的解旋力，并稳定了一种类似六体的中间体，该中间体在连续的解旋循环中保留了两个标记的二聚体。转录后也观察到了这种中间体。总之，FACT通过削弱屏障并在酶通过后积极协助维持核小体完整性来促进核小体转录。
