CEITEC-Central European Institute of Technology, Masaryk University, Brno CZ-62500, Czech Republic.
National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno CZ-62500, Czech Republic.
Nucleic Acids Res. 2022 Jun 10;50(10):5961-5973. doi: 10.1093/nar/gkac451.
Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6's function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.
转录延伸因子 Spt6 与 RNA 聚合酶 II（Pol II）结合，并作为组蛋白伴侣发挥作用，促进 Pol II 通过后核小体的重新组装。Spt6 介导的核小体重新组装的确切机制仍不清楚。在这项研究中，我们使用了一种结合冷冻电子显微镜和小角 X 射线散射的杂交方法来可视化酿酒酵母 Spt6 的结构。重建的 Spt6 整体结构不仅揭示了 Spt6 的核心结构，还揭示了其灵活的 N 端和 C 端，这对 Spt6 的功能至关重要。我们发现 Spt6 的酸性 N 端区域不仅防止 Spt6 与 Pol II CTD 和 Pol II CTD-接头结合，还防止与预形成的完整核小体和核小体 DNA 结合。Spt6 的 N 端区域与 tSH2 结构域和 Spt6 的核心自缔合，从而控制与 Pol II 和核小体的结合。此外，我们发现 Spt6 促进体外核小体的组装。这些数据表明，Spt6 中固有无序和结构区域之间的合作调节核小体和 Pol II CTD 的结合，以及核小体的组装。
