Molecular mechanisms of substrate-controlled ring dynamics and sub-stepping in a nucleic-acid dependent hexameric motor

Abstract

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.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).Structural studies have provided insights into the mechanics of RNA...

Ligand-induced and small-molecule control of substrate loading in a hexameric helicase

“…4B), showing that the secondary site becomes capable of stably binding RNA as the Rho ring closes. These findings are consistent with the SAXS studies carried out in the accompanying study (16), showing that both RNA and nucleotide are needed to promote ring closure cooperatively in Rho.…”

“…2A), nucleotide-dependent differences in curve shapes were clearly evident, similar to those reported in the accompanying study ( Fig. S3) (16). Visual inspection of the curves over a q range from 0.07-0.13 Å −1 , the region of the curve in which nucleotide-dependent differences are most pronounced ( Fig.…”

“…To determine the impact of bicyclomycin on the Rho conformational state, we first used SAXS to monitor structural dynamics in solution directly. An accompanying study demonstrates that open-ring Rho molecules close in the presence of RNA and the nonhydrolyzable ATP analog ADP·BeF 3 but not in the presence of either ligand individually (16). Because bicyclomycin is a relatively modest inhibitor of Rho ATPase activity (IC 50 = 20 μM) (25), we reasoned that its effects on ring closure might be most evident when examined over a range of nucleotide concentrations.…”

“…Binding of pyrimidine-rich sequences to the N-terminal RNA-binding domains of Rho is a particularly well-known accelerant of Rho's ATPase activity (30), with presteady-state ATPase assays showing that the formation of a catalytically competent Rho ring is governed by a rate-limiting RNA-and ATP-dependent conformational change (8). The liganddependence of this isomerization event correlates with the requirements for ring closure identified in an accompanying study (16). These findings raise the intriguing possibility that the sequence specificity of Rho-dependent termination is caused in part by an increased efficiency of ring closure when the N-terminal RNA-binding domains are occupied by pyrimidine-rich sequences.…”

“…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), rearranges residues in the Rho ATPbinding pockets into a hydrolysis-competent state (17). Once engaged, Rho maintains primary-site contacts with the rut sequence as it translocates 5′ to 3′ along the RNA strand in an ATP-dependent manner, a process known as "tethered tracking" (18)(19)(20)(21).…”