Key Laboratory of Synthetic Biology, State Key Laboratory of Plant Design, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Sci Adv. 2024 Aug 23;10(34):eadq3087. doi: 10.1126/sciadv.adq3087.
RNA polymerase IV (Pol IV) forms a complex with RNA-directed RNA polymerase 2 (RDR2) to produce double-stranded RNA (dsRNA) precursors essential for plant gene silencing. In the "backtracking-triggered RNA channeling" model, Pol IV backtracks and delivers its transcript's 3' terminus to RDR2, which synthesizes dsRNA. However, the mechanisms underlying Pol IV backtracking and RNA protection from cleavage are unclear. Here, we determined cryo-electron microscopy structures of Pol IV elongation complexes at four states of its nucleotide addition cycle (NAC): posttranslocation, guanosine triphosphate-bound, pretranslocation, and backtracked states. The structures reveal that Pol IV maintains an open DNA cleft and kinked bridge helix in all NAC states, loosely interacts with the nucleoside triphosphate substrate, and barely contacts proximal backtracked nucleotides. Biochemical data indicate that Pol IV is inefficient in forward translocation and RNA cleavage. These findings suggest that Pol IV transcription elongation is prone to backtracking and incapable of RNA hydrolysis, ensuring efficient dsRNA production by Pol IV-RDR2.
RNA 聚合酶 IV（Pol IV）与 RNA 指导的 RNA 聚合酶 2（RDR2）形成复合物，以产生双链 RNA（dsRNA）前体，这些前体对植物基因沉默至关重要。在“回溯触发的 RNA 沟道”模型中，Pol IV 回溯并将其转录物的 3'末端递送到 RDR2，后者合成 dsRNA。然而，Pol IV 回溯和 RNA 免受切割的保护机制尚不清楚。在这里，我们确定了 Pol IV 延伸复合物在其核苷酸添加循环（NAC）的四个状态下的低温电子显微镜结构：易位后、鸟苷三磷酸结合、易位前和回溯状态。这些结构表明，Pol IV 在所有 NAC 状态下均保持开放的 DNA 裂缝和扭曲的桥螺旋，与核苷三磷酸底物松散相互作用，并且几乎不接触近端回溯的核苷酸。生化数据表明，Pol IV 在正向易位和 RNA 切割方面效率低下。这些发现表明，Pol IV 转录延伸容易回溯并且不能进行 RNA 水解，从而确保 Pol IV-RDR2 高效产生 dsRNA。
