Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

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