Binding and translocation of termination factor Rho studied at the single-molecule level
Koslover DJ*, Fazal FM*, Mooney RA, Landick R, Block SM.
Journal of Molecular Biology (JMB), 2012
Rho 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 ฮปtR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ยฑ 2nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ยฑ 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'โ3' 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.
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