Transcription of transfer RNA (tRNA) genes by RNA polymerase (Pol) III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) to reveal structural and real-time kinetic insights into how the 520-kDa yeast TFIIIC complex engages A-box and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of τA and τB subcomplexes bound to the A-box and B-box were obtained at 3.7 and 2.5 Å resolution, respectively, while cryo-EM single-particle mapping determined the specific distance and relative orientation of the τA and τB subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of τA and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters.

中文翻译：

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酵母一般转录因子 TFIIIC 对 tRNA 启动子参与的结构和动力学见解


RNA 聚合酶 （Pol） III 转录转移 RNA （tRNA） 基因需要一般转录因子 IIIC （TFIIIC），它识别 II 型基因启动子的基因内 A-box 和 B-box DNA 基序。然而，其潜在机制仍然难以捉摸，部分原因是缺少用于 A-box 识别的结构信息。在这项研究中，我们使用单颗粒低温电子显微镜 （cryo-EM） 和单分子荧光共振能量转移 （smFRET） 来揭示 520-kDa 酵母 TFIIIC 复合物如何在 tRNA 基因启动子的背景下与 A-box 和 B-box DNA 基序结合的结构和实时动力学见解。与 A-box 和 B-box 结合的 τA 和 τB 亚复合物的冷冻电镜结构分别以 3.7 和 2.5 Å 的分辨率获得，而冷冻电镜单粒子映射确定了 τA 和 τB 子复合物的比距离和相对方向，揭示了 TFIIIC 的完全参与状态。smFRET 实验表明，TFIIIC 在 tRNA 基因上的总体募集和停留时间主要受 B-box 识别控制，而足迹实验表明，在 TFIIIC 识别 II 型启动子后，τA 和 A-box 在 TFIIIB 和 Pol III 募集中起关键作用。