NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription.

Ring-shaped hexameric helicases and translocases support essential DNA-, RNA-, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the Escherichia coli Rho transcription termination factor as a model system, we have used solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. Small-angle X-ray scattering data show that Rho preferentially adopts an open-ring state in solution and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish structural snapshots of substepping events in a hexameric helicase/translocase.ATPase | helicase | motor protein | transcription | translocase H exameric helicases and translocases are motor proteins that play a central role in cellular transactions ranging from replication and repair to transcriptional regulation, chromosome packaging, and proteolytic homeostasis (1-4). Used to drive the processive and, at times, highly rapid movement of extended nucleic acid or protein chains through a central pore, ring-shaped motors face several challenges to their operation. One is that certain enzymes must transition through controlled ring opening and/or subunit assembly events to allow long, polymeric substrates that lack freely accessible ends to access interior motor elements (5-7). Another is that, once loaded, the molecular plasticity inherent to these assemblies must be harnessed to precisely coordinate ATP binding and hydrolysis between multiple subunits with the powering of substrate translocation, while at the same time alternating between tight and loose grips on the substrate to allow for processive movement. The substrate-dependent molecular rearrangements that underpin ring dynamics during these events remain poorly understood, not only for hexameric helicases and translocases, but for related ring-shaped switches as well.The Escherichia coli Rho transcription termination factor is a well-established model system for understanding hexameric translocase and helicase function (8, 9). During termination, Rho uses a cytosine-specific RNA-binding domain appended to the N terminus of a RecA-type ATPase fold (10, 11) to bind nascent RNA transcripts at cytosine-rich sequences [known as Rho utilization (rut) sites] (12, 13). Once loaded, Rho consumes ATP to translocate 5′→3′ toward a paused RNA polymerase, eventually promoting transcription bubble collapse and RNA release (14-19).Str...

Processive, ring-shaped protein and nucleic acid protein translocases control essential biochemical processes throughout biology and are considered high-prospect therapeutic targets. The Escherichia coli Rho factor is an exemplar hexameric RNA translocase that terminates transcription in bacteria. As with many ring-shaped motor proteins, Rho activity is modulated by a variety of poorly understood mechanisms, including small-molecule therapeutics, protein-protein interactions, and the sequence of its translocation substrate. Here, we establish the mechanism of action of two Rho effectors, the antibiotic bicyclomycin and nucleic acids that bind to Rho's primary RNA recruitment site. Using small-angle X-ray scattering and a fluorescence-based assay to monitor the ability of Rho to switch between open-ring (RNA-loading) and closed-ring (RNA-translocation) states, we found bicyclomycin to be a direct antagonist of ring closure. Reciprocally, the binding of nucleic acids to its N-terminal RNA recruitment domains is shown to promote the formation of a closed-ring Rho state, with increasing primary-site occupancy providing additive stimulatory effects. This study establishes bicyclomycin as a conformational inhibitor of Rho ring dynamics, highlighting the utility of developing assays that read out protein conformation as a prospective screening tool for ring-ATPase inhibitors. Our findings further show that the RNA sequence specificity used for guiding Rho-dependent termination derives in part from an intrinsic ability of the motor to couple the recognition of pyrimidine patterns in nascent transcripts to RNA loading and activity.antibiotic | ATPase | helicase | motor protein | transcription R ing-shaped hexameric helicases and translocases are motor proteins that control myriad essential viral and cellular processes. Many hexameric motors undergo substrate-dependent conformational changes that couple activity to the productive binding of client substrates (1-4). Internal regulatory domains and exogenous proteins or small molecules frequently impact client substrate recruitment and engagement by these enzymes (5-8); however, it is generally unclear how such factors control helicase or translocase dynamics.Rho is a hexameric helicase responsible for controlling ∼20% of all transcription termination events in Escherichia coli (9). Rho is initially recruited to nascent transcripts in an open, lock washer-shaped configuration (Fig. 1A) (10, 11), where it binds preferentially to pyrimidine-rich sequences (termed "Rho utilization of termination" sequences, or "rut" sites) using a primary RNA-binding site located in the N-terminal OB folds of the hexamer (12-14). Following rut recognition, Rho converts into a closed-ring form (Fig. 1B), locking the RNA strand into a secondary RNA-binding site formed by two conserved sequence elements known as the "Q" and "R" loops (15) located within the central pore of the hexamer. This conformational change, which we show in an accompanying paper to be both RNAand ATP-dependent (16), rearran...

The bacterial hexameric helicase known as Rho is an archetypal sequence-specific transcription terminator that typically halts the synthesis of a defined set of transcripts, particularly those bearing cytosine-rich 3' untranslated regions. However, under conditions of translational stress, Rho can also terminate transcription at cytosine-poor sites when assisted by the transcription factor NusG. Recent structural, biochemical, and computational studies of the Rho•NusG interaction in Escherichia coli have helped establish how NusG reprograms Rho activity. NusG is found to be an allosteric activator of Rho that directly binds to the ATPase motor domain of the helicase and facilitates closure of the Rho ring around non-ideal (purine-rich) target RNAs. The manner in which NusG acts on Rho helps to explain how the transcription terminator is excluded from acting on RNA polymerase by exogenous factors, such as the antitermination protein NusE, the NusG paralog RfaH, and RNA polymerase-coupled ribosomes. Collectively, an understanding of the link between NusG and Rho provides new insights into how transcriptional and translational fidelity are maintained during gene expression in bacteria.