Binding and Translocation ofTermination Factor RhoStudiedat the Single-Molecule Level

Daniel J. Koslover, Furqan M. Fazal, Rachel A. Mooney, Robert Landick, Steven M. Block

Rho termination factor is an essential hexameric helicase responsible for terminating 20–50% of all mRNA synthesis in E. coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho-utilization (rut) site of the λtR1 terminator. Our results are consistent with Rhocomplexes adopting two states, one that binds 57 ± 2 nucleotides of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2 nucleotides at the six primary sitesplus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′-to-3′ towards RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the rut site and RNAP. These findings lead to a general model forRho binding and translocation, and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins.

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