Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ

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.