Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, Republic of China.
Mol Cell Biol. 2011 Jul;31(13):2715-28. doi: 10.1128/MCB.05151-11. Epub 2011 May 2.
Eukaryotic RNA polymerase III (Pol III) relies on a transcription factor TFIIF-like Rpc37/53 subcomplex for promoter opening, elongation, termination, and reinitiation. By incorporating the photoreactive amino acid p-benzoyl-L-phenylalanine (BPA) into Rpc37, Rpc53, and the Rpc2 subunit of Pol III, we mapped protein-protein interactions, revealing the position of Rpc37/53 within the Pol III preinitiation complex (PIC). BPA photo-cross-linking was combined with site-directed hydroxyl radical probing to localize the Rpc37/53 dimerization module on the lobe/external 2 domains of Rpc2, in similarity to the binding of TFIIF on Pol II. N terminal to the dimerization domain, Rpc53 binds the Pol III-specific subunits Rpc82 and Rpc34, the Pol III stalk, and the assembly factor TFIIIC, essential for PIC formation. The C-terminal domain of Rpc37 interacts extensively with Rpc2 and Rpc34 and contains binding sites for initiation factor Bdp1. We also located the C-terminal domain of Rpc37 within the Pol III active center in the ternary elongation complex, where it likely functions in accurate termination. Our work explains how the Rpc37/53 dimer is anchored on the Pol III core and acts as a hub to integrate a protein network for initiation and termination.
真核 RNA 聚合酶 III（Pol III）依赖转录因子 TFIIF 样 Rpc37/53 亚基复合物来启动、延伸、终止和重新起始转录。通过将光反应性氨基酸对苯甲酰-L-苯丙氨酸（BPA）整合到 Rpc37、Rpc53 和 Pol III 的 Rpc2 亚基中，我们绘制了蛋白质-蛋白质相互作用图谱，揭示了 Rpc37/53 在 Pol III 起始前复合物（PIC）中的位置。BPA 光交联与定点羟自由基探测相结合，将 Rpc37/53 二聚体模块定位在 Rpc2 的叶/外部 2 结构域上，类似于 TFIIF 在 Pol II 上的结合。在二聚体结构域的 N 端，Rpc53 结合 Pol III 特异性亚基 Rpc82 和 Rpc34、Pol III 柄部和组装因子 TFIIIC，这对于 PIC 形成是必不可少的。Rpc37 的 C 端结构域与 Rpc2 和 Rpc34 广泛相互作用，并包含起始因子 Bdp1 的结合位点。我们还在三元延伸复合物中的 Pol III 活性中心定位了 Rpc37 的 C 端结构域，它可能在准确终止中发挥作用。我们的工作解释了 Rpc37/53 二聚体如何锚定在 Pol III 核心上，并作为一个枢纽，整合起始和终止的蛋白质网络。
