Wang Qianmin, Ren Yulei, Jin Qianwei, Chen Xizi, Xu Yanhui
Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, New Cornerstone Science Laboratory, State Key Laboratory of Genetics and Development of Complex Phenotypes, Department of Biochemistry and Biophysics, School of Life Sciences, Shanghai Key Laboratory of Radiation Oncology and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College of Fudan University, Shanghai, China.
Sycamore Research Institute of Life Sciences, Shanghai, China.
Nature. 2025 Jun 4. doi: 10.1038/s41586-025-09093-w.
RNA polymerase III (Pol III) transcribes highly demanded RNAs grouped into three types of classical promoters, including type 1 (5S rRNA), type 2 (tRNA) and type 3 (short non-coding RNAs, such as U6, 7SK and RNase H1) promoters. While structures of the Pol III preinitiation complex (PIC) and elongation complex (EC) have been determined, the mechanism underlying the transition from initiation to elongation remains unclear. Here we reconstituted seven human Pol III transcribing complexes (TC4, TC5, TC6, TC8, TC10, TC12 and TC13) halted on U6 promoters with nascent RNAs of 4-13 nucleotides. Cryo-electron microscopy structures captured initially transcribing complexes (ITCs; TC4 and TC5) and ECs (TC6-13). Together with KMnO footprinting, the data reveal extensive modular rearrangements: the transcription bubble expands from PIC to TC5, followed by general transcription factor (GTF) dissociation and abrupt bubble collapse from TC5 to TC6, marking the ITC-EC transition. In TC5, SNAPc and TFIIIB remain bound to the promoter and Pol III, while the RNA-DNA hybrid adopts a tilted conformation with template DNA blocked by BRF2, a TFIIIB subunit. Hybrid forward translocation during ITC-EC transition triggers BRF2-finger retraction, GTF release and transcription-bubble collapse. Pol III then escapes the promoter while GTFs stay bound upstream, potentially enabling reinitiation. These findings reveal molecular insights into Pol III dynamics and reinitiation mechanisms on type 3 promoters of highly demanded small RNAs, with the earliest documented initiation-elongation transition for an RNA polymerase.
RNA聚合酶III（Pol III）转录高度必需的RNA，这些RNA分为三种经典启动子类型，包括1型（5S rRNA）、2型（tRNA）和3型（短非编码RNA，如U6、7SK和核糖核酸酶H1）启动子。虽然已经确定了Pol III起始前复合物（PIC）和延伸复合物（EC）的结构，但从起始到延伸的潜在机制仍不清楚。在这里，我们重建了七种在U6启动子上停滞的人类Pol III转录复合物（TC4、TC5、TC6、TC8、TC10、TC12和TC13），其新生RNA长度为4至13个核苷酸。冷冻电子显微镜结构捕捉到了初始转录复合物（ITC；TC4和TC5）和延伸复合物（TC6 - 13）。结合高锰酸钾足迹法，数据揭示了广泛的模块重排：转录泡从PIC扩展到TC5，随后通用转录因子（GTF）解离，转录泡从TC5到TC6突然塌陷，标志着ITC - EC转变。在TC5中，SNAPc和TFIIIB仍然与启动子和Pol III结合，而RNA - DNA杂交体采用倾斜构象，模板DNA被TFIIIB亚基BRF2阻断。ITC - EC转变过程中的杂交体向前移位触发BRF2手指缩回、GTF释放和转录泡塌陷。然后Pol III逃离启动子，而GTF仍结合在上游，这可能使重新起始成为可能。这些发现揭示了对高度必需的小RNA的3型启动子上Pol III动态和重新起始机制的分子见解，这是RNA聚合酶最早记录的起始 - 延伸转变。
