Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, United States.
Department of Chemistry, Washington University in St. Louis, One Brookings Drive, St. Louis, MO 63130, United States.
J Mol Biol. 2018 Mar 2;430(5):641-654. doi: 10.1016/j.jmb.2018.01.003. Epub 2018 Jan 31.
RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre-initiation complex. Upstream Activating Factor (UAF) is one of two multi-subunit complexes that regulate pre-initiation complex formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. We found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3-H4 tetramer-like subcomplex.
酿酒酵母 RNA 聚合酶 I（Pol I）转录需要四个独立的因子，这些因子将 Pol I 招募到启动子上，形成起始前复合物。上游激活因子（UAF）是两个多亚基复合物之一，通过与核糖体 DNA 启动子结合并刺激下游 Pol I 因子的招募来调节起始前复合物的形成。UAF 由 Rrn9、Rrn5、Rrn10、Uaf30 和组蛋白 H3 和 H4 组成。我们开发了一种基于重组大肠杆菌的系统，用于共表达和纯化具有转录活性的 UAF 复合物，并研究每个亚基在复合物形成中的重要性。我们发现，没有一个单一的亚基是 UAF 组装所必需的，包括组蛋白 H3 和 H4。我们还证明组蛋白 H3 能够与每个 UAF 特异性亚基相互作用，并表明该复合物中存在至少两个组蛋白 H3 和一个 H4 拷贝。总之，我们的结果提供了一个新的模型，表明 UAF 包含一个混合的 H3-H4 四聚体样亚基。
