Ligand-induced and small-molecule control of substrate loading in a hexameric helicase.
Processive, ring-shaped protein and nucleic acid protein translocases control essential biochemical processes throughout biology and are considered high-prospect therapeutic targets. The Escherichia coli Rho factor is an exemplar hexameric RNA translocase that terminates transcription in bacteria. As with many ring-shaped motor proteins, Rho activity is modulated by a variety of poorly understood mechanisms, including small-molecule therapeutics, protein-protein interactions, and the sequence of its translocation substrate. Here, we establish the mechanism of action of two Rho effectors, the antibiotic bicyclomycin and nucleic acids that bind to Rho's primary RNA recruitment site. Using small-angle X-ray scattering and a fluorescence-based assay to monitor the ability of Rho to switch between open-ring (RNA-loading) and closed-ring (RNA-translocation) states, we found bicyclomycin to be a direct antagonist of ring closure. Reciprocally, the binding of nucleic acids to its N-terminal RNA recruitment domains is shown to promote the formation of a closed-ring Rho state, with increasing primary-site occupancy providing additive stimulatory effects. This study establishes bicyclomycin as a conformational inhibitor of Rho ring dynamics, highlighting the utility of developing assays that read out protein conformation as a prospective screening tool for ring-ATPase inhibitors. Our findings further show that the RNA sequence specificity used for guiding Rho-dependent termination derives in part from an intrinsic ability of the motor to couple the recognition of pyrimidine patterns in nascent transcripts to RNA loading and activity.
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
Michael R
MR
Lawson
Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
Kevin
K
Dyer
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; jmberger@jhmi.edu.|Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
James M
JM
Berger
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008024:Ligands; D008958:Models, Molecular; D010458:Peptidyl Transferases / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D020365:RNA Helicases / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D012234:Rho Factor / Q000737:chemistry* / Q000235:genetics; D014158:Transcription, Genetic
D008024:Ligands; D016601:RNA-Binding Proteins; D012234:Rho Factor; D012313:RNA; D010458:Peptidyl Transferases; D000251:Adenosine Triphosphatases; D020365:RNA Helicases
ATPase; antibiotic; helicase; motor protein; transcription