How to stop RNA polymerase Timely and tunable cessation of RNA synthesis is vital for cellular homeostasis. RNA helicases such as the archetypal termination factor r actively dismantle transcription complexes, but the transitory nature of termination makes the process hard to study structurally. Said et al. assembled ρ-bound transcription complexes and studied them using cryo–electron microscopy with an approach that captured a series of functional states en route to termination. They found an extensive and dynamic network of r interactions with RNA polymerase, nucleic acids, and accessory Nus factors. ρ mediates stepwise rearrangements of these contacts, transforming an actively transcribing complex into a moribund pretermination intermediate. Science, this issue p. 44 Structure/function analyses reveal how termination factor ρ captures and inactivates a transcription elongation complex. INTRODUCTION Factor-dependent transcription termination is essential to limit pervasive transcription, maintain genome stability, balance the expression of neighboring genes, and recycle RNA polymerase (RNAP). Two main classes of models can explain how termination factors stop RNA synthesis. In RNA-centric models, a terminator, powered by adenosine triphosphate (ATP)–dependent RNA translocase activity or by exonucleolytic RNA degradation, moves along the nascent RNA and rear-ends RNAP, dislodging it from the RNA. In transcription elongation complex (EC)–centric models, a terminator induces conformational changes in RNAP that inactivate it. Evidence in support of both mechanisms exists for translocases and exonucleases that elicit termination in bacteria and eukaryotes, but molecular details of their actions remain elusive because, once committed to termination, transcription complexes disassemble rapidly and are thus refractory to structure/function analyses. RATIONALE To elucidate the structural basis for termination, we used the archetypal ring-shaped hexameric helicase ρ. Escherichia coli ρ, perhaps the strongest molecular motor known, can load onto free RNA as an open ring, close the ring around the RNA, and engage in ATP-dependent translocation, removing any obstacle from its path. During transcription, ρ triggers RNA release from the EC within a well-defined termination zone once ~90 nucleotides of C-rich RNA, which ρ binds with high affinity, have been synthesized by RNAP. We surmised that a ρ-bound EC poised to enter this termination zone will be metastable, giving rise to an ensemble of intermediates en route to termination. We used single-particle cryo–electron microscopy (cryo-EM) to analyze these “peri-termination” E. coli ECs bound to ρ, an ATP analog, and general elongation factors NusA and NusG known to modulate ρ activity. We also carried out in vitro and in vivo functional assays to validate key interactions suggested by our structural analysis. RESULTS We report the structures of seven intermediates along the termination pathway. ρ is recruited to the EC via extensive contacts to RNAP, NusA, and NusG, but initially makes no contacts with RNA. After recruitment, rearrangements of the ρ hexamer, NusA, upstream DNA, and several regions of RNAP set up a stage for RNA engagement by ρ. The N-terminal zinc-binding domain of the RNAP β′ subunit aids ρ in capturing the nascent RNA, a synergy that is supported by in vivo analysis of ρ and β′ mutants. Upon anchoring the RNA, ρ induces structural rearrangements that lead to the displacement of NusG and weakening of the RNAP grip on nucleic acids due to partial opening of the β′ clamp domain. The formation of a moribund complex, in which the clamp is wide open and the RNA is dislodged from the active site, completes the RNAP inactivation by ρ. Remarkably, the ρ ring is held open by the network of ρ contacts with RNAP and NusA throughout the entire pathway, preventing ρ from exerting force on RNA. Our data argue that ρ travels with RNAP rather than chases after it, and that termination is favored by pause-promoting conformational changes in the EC rather than by the reduced rate of RNA synthesis. CONCLUSION This study explains how ρ is targeted to RNAs that are still being made and cooperates with NusA and NusG to effect striking conformational changes that inactivate the transcribing RNAP. Hitchhiking on RNAP enables ρ to survey and silence useless and harmful transcripts independently of their sequence, as documented for several bacterial ρ orthologs. Unexpectedly, ρ stalls transcription without engaging its powerful motor activity, which may be essential after termination to destroy R-loops, the toxic by-products of the EC dissociation. A growing list of allosteric mechanisms of transcription regulation suggests that many accessory factors may exploit dynamic properties of RNAP to modulate RNA synthesis, acting together with the orthologs/analogs of Nus factors present in all domains of life. ρ traps NusA/NusG-modified elongation complexes in a moribund state. NusA and NusG are the only general transcription factors in E. coli that modulate ρ-dependent termination. Conflicting models explain how ρ terminates RNA synthesis. Cryo-EM analysis of ρ/NusA/NusG-ECs and structure-informed biochemical analyses support an EC-centric model, revealing how an initial engagement complex is converted stepwise to a moribund complex. The pathway involves rearrangements of ρ, NusA/G, and RNAP elements, and culminates in a massive displacement of the RNA 3′-end from the RNAP active site. Factor-dependent transcription termination mechanisms are poorly understood. We determined a series of cryo–electron microscopy structures portraying the hexameric adenosine triphosphatase (ATPase) ρ on a pathway to terminating NusA/NusG-modified elongation complexes. An open ρ ring contacts NusA, NusG, and multiple regions of RNA polymerase, trapping and locally unwinding proximal upstream DNA. NusA wedges into the ρ ring, initially sequestering RNA. Upon deflection of distal upstream DNA over the RNA polymerase zinc-binding domain, NusA rotates underneath one capping ρ subunit, which subsequently captures RNA. After detachment of NusG and clamp opening, RNA polymerase loses its grip on the RNA:DNA hybrid and is inactivated. Our structural and functional analyses suggest that ρ, and other termination factors across life, may use analogous strategies to allosterically trap transcription complexes in a moribund state.

中文翻译：

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通过终止子 ATPase ρ 使非易位的 RNA 聚合酶失活的步骤

如何停止 RNA 聚合酶 及时、可调整地停止 RNA 合成对于细胞稳态至关重要。RNA 解旋酶，如原型终止因子 r 会主动拆除转录复合物，但终止的短暂性质使得该过程难以在结构上进行研究。赛义德等人。组装 ρ 结合的转录复合物，并使用冷冻电子显微镜研究它们，其方法是在终止过程中捕获一系列功能状态。他们发现了 r 与 RNA 聚合酶、核酸和辅助 Nus 因子相互作用的广泛而动态的网络。ρ 介导这些接触的逐步重排，将主动转录的复合物转化为垂死的预终止中间体。科学，这个问题 p。44 结构/功能分析揭示了终止因子 ρ 如何捕获和灭活转录延伸复合物。引言 因子依赖性转录终止对于限制普遍转录、维持基因组稳定性、平衡邻近基因的表达和循环 RNA 聚合酶 (RNAP) 至关重要。两大类模型可以解释终止因子如何阻止 RNA 合成。在以 RNA 为中心的模型中，由依赖三磷酸腺苷 (ATP) 的 RNA 转位酶活性或外切核酸酶降解的终止子沿着新生的 RNA 移动并后端 RNAP，将其从 RNA 中移出。在以转录延伸复合体 (EC) 为中心的模型中，终止子诱导 RNAP 的构象变化，使其失活。对于在细菌和真核生物中引发终止的易位酶和外切核酸酶，存在支持这两种机制的证据，但它们作用的分子细节仍然难以捉摸，因为一旦终止，转录复合物会迅速分解，因此难以进行结构/功能分析。基本原理为了阐明终止的结构基础，我们使用了原型环状六聚解旋酶 ρ。大肠杆菌 ρ 可能是已知最强的分子马达，它可以作为开环加载到游离 RNA 上，关闭 RNA 周围的环，并参与 ATP 依赖性易位，消除其路径上的任何障碍。在转录过程中，一旦 ρ 以高亲和力结合的富含 C 的 RNA 的约 90 个核苷酸被 RNAP 合成，ρ 就会在明确定义的终止区内触发 RNA 从 EC 释放。我们推测准备进入该终止区的 ρ 结合 EC 将是亚稳态的，从而在终止的途中产生中间体集合。我们使用单粒子冷冻电子显微镜 (cryo-EM) 来分析这些与 ρ（一种 ATP 类似物）以及已知可调节 ρ 活性的一般延伸因子 NusA 和 NusG 结合的“周围终止”大肠杆菌 EC。我们还进行了体外和体内功能分析，以验证我们的结构分析建议的关键相互作用。结果我们报告了终止途径中七个中间体的结构。ρ 通过与 RNAP、NusA 和 NusG 的广泛接触被招募到 EC，但最初不与 RNA 接触。招募后，ρ 六聚体、NusA、上游 DNA 和 RNAP 的几个区域的重排为 ρ 的 RNA 参与建立了一个阶段。RNAP β' 亚基的 N 端锌结合域有助于 ρ 捕获新生的 RNA，这种协同作用得到了 ρ 和 β' 突变体的体内分析的支持。在锚定 RNA 后，ρ 诱导结构重排，导致 NusG 的置换和 RNAP 对核酸的抓地力减弱，这是由于 β' 钳结构域的部分开放。垂死的复合物的形成，其中夹子是敞开的，RNA 从活性位点上脱落，通过 ρ 完成了 RNAP 的失活。值得注意的是，ρ 环在整个通路中由 ρ 与 RNAP 和 NusA 接触的网络保持打开状态，防止 ρ 对 RNA 施加力。我们的数据表明 ρ 与 RNAP 一起传播而不是追逐它，并且这种终止有利于暂停促进 EC 中的构象变化，而不是降低 RNA 合成率。结论 本研究解释了 ρ 如何靶向仍在制造的 RNA，并与 NusA 和 NusG 合作以实现显着的构象变化，从而使转录的 RNAP 失活。在 RNAP 上搭便车使 ρ 能够独立于其序列调查和沉默无用和有害的转录本，如几个细菌 ρ 直向同源物所记录的那样。出乎意料的是，ρ 在不参与其强大的运动活动的情况下停止转录，这可能在终止后破坏 R 环（EC 解离的有毒副产物）是必不可少的。越来越多的转录调控变构机制表明，许多辅助因子可能利用 RNAP 的动态特性来调节 RNA 合成，与生活的所有领域中存在的 Nus 因子的直系同源物/类似物一起起作用。ρ 在垂死状态下捕获 NusA/NusG 修饰的伸长复合物。NusA 和 NusG 是大肠杆菌中唯一调节 ρ 依赖性终止的通用转录因子。相互矛盾的模型解释了 ρ 如何终止 RNA 合成。ρ/NusA/NusG-ECs 的 Cryo-EM 分析和基于结构的生化分析支持以 EC 为中心的模型，揭示了初始参与复合体如何逐步转换为垂死的复合体。该途径涉及 ρ、NusA/G 和 RNAP 元件的重排，最终导致 RNA 3'-末端从 RNAP 活性位点大量置换。因子依赖性转录终止机制知之甚少。我们确定了一系列冷冻电子显微镜结构，描绘了终止 NusA/NusG 修饰的延伸复合物的途径上的六聚腺苷三磷酸酶 (ATPase) ρ。一个开放的 ρ 环接触 NusA、NusG 和 RNA 聚合酶的多个区域，捕获和局部展开近端上游 DNA。NusA 楔入 ρ 环，最初隔离 RNA。当远端上游 DNA 在 RNA 聚合酶锌结合域上偏转时，NusA 在一个加帽 ρ 亚基下方旋转，随后捕获 RNA。在 NusG 分离并打开钳夹后，RNA 聚合酶失去对 RNA:DNA 杂交体的控制并失活。我们的结构和功能分析表明，ρ 和其他终生终止因子可能会使用类似的策略以变构方式捕获处于垂死状态的转录复合物。