PT  - JOURNAL ARTICLE
AU  - Nathan D. Thomsen
AU  - Michael R. Lawson
AU  - Lea B. Witkowsky
AU  - Song Qu
AU  - James M. Berger
TI  - Molecular mechanisms of substrate-controlled ring dynamics and sub-stepping in a nucleic-acid dependent hexameric motor
AID  - 10.1101/069559
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 069559
4099  - http://biorxiv.org/content/early/2016/08/16/069559.short
4100  - http://biorxiv.org/content/early/2016/08/16/069559.full
AB  - 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 E. coli Rho transcription termination factor as a model system, we have employed solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. SAXS 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 the first structural snapshots of substepping events in a hexameric helicase/translocase.SIGNIFICANCE Hexameric, ring-shaped translocases are molecular motors that convert the chemical energy of ATP hydrolysis into the physical movement of protein and nucleic acid substrates. Structural studies of several distinct hexameric translocases have provided insights into how substrates are loaded and translocated; however, the range of structural changes required for coupling ATP turnover to a full cycle of substrate loading and translocation has not been visualized for any one system. Here, we combine low-and high-resolution structural studies of the Rho helicase, defining for the first time the ensemble of conformational transitions required both for substrate loading in solution and for substrate movement by a processive hexameric translocase.