Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA; Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, USA.
Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.
Mol Cell. 2023 Aug 3;83(15):2641-2652.e7. doi: 10.1016/j.molcel.2023.06.015. Epub 2023 Jul 3.
RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.
RNA 聚合酶 III（Pol III）负责转录 5S 核糖体 RNA（5S rRNA）、tRNA 和其他短非编码 RNA。其招募到 5S rRNA 启动子需要转录因子 TFIIIA、TFIIIC 和 TFIIIB。在这里，我们使用冷冻电子显微镜（cryo-EM）可视化结合到启动子上的 S. cerevisiae TFIIIA 和 TFIIIC 复合物。基因特异性因子 TFIIIA 与 DNA 相互作用，并作为 TFIIIC-启动子相互作用的接头。我们还观察到 TFIIIB 亚基 Brf1 和 TBP（TATA 盒结合蛋白）的 DNA 结合，导致全长 5S rRNA 基因缠绕在复合物周围。我们的 smFRET 研究表明，复合物内的 DNA 在慢时间尺度上经历了剧烈弯曲和部分解离，与我们的 cryo-EM 结果预测的模型一致。我们的发现为 5S rRNA 启动子上的转录起始复合物组装提供了新的见解，并允许我们直接比较 Pol III 和 Pol II 转录适应。
