Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C.
Institute of Chemistry, Academia Sinica, Taiwan, R.O.C.
Nucleic Acids Res. 2023 May 22;51(9):4223-4236. doi: 10.1093/nar/gkac1163.
Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse. We conducted a mutational analysis on Rpc31, which uncovered a functional peptide adjacent to the highly conserved Asp-Glu-rich acidic C-terminus. This C-terminal peptide region, termed 'pre-acidic', is important for optimal cell growth, tRNA synthesis, and stable association of Rpc31 in the pre-initiation complex (PIC). Our site-directed photo-cross-linking to map protein interactions within the PIC reveal that this pre-acidic region specifically targets Rpc34 during transcription initiation, but also interacts with the DNA entry surface in free pol III. Thus, we have uncovered a switchable Rpc31 C-terminal region that functions in an initiation-specific protein interaction for pol III transcription.
Rpc31 是酿酒酵母 RNA 聚合酶 III（pol III）中 TFIIE 相关 Rpc82/34/31 异三聚体亚基复合物的一个亚基。对 pol III 的结构分析表明，Rpc31 的 N 端区域锚定于 Rpc82 上，并进一步与聚合酶核心和柄亚基相互作用。然而，Rpc31 的 C 端区域的结构和功能信息却很少。我们对 Rpc31 进行了突变分析，结果发现一个紧邻高度保守的天冬氨酸-谷氨酸丰富酸性 C 端的功能肽。这个 C 端肽区域，称为“前酸性”，对于最优的细胞生长、tRNA 合成以及在起始前复合物（PIC）中 Rpc31 的稳定结合是重要的。我们通过定点光交联来绘制 PIC 内的蛋白质相互作用图谱，结果表明这个前酸性区域在转录起始时特异性靶向 Rpc34，但也与游离 pol III 中的 DNA 进入表面相互作用。因此，我们发现了一个可切换的 Rpc31 C 端区域，它在 pol III 转录的起始特异性蛋白质相互作用中发挥作用。
