期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：48
页码：13714-13719
DOI：10.1073/pnas.1616749113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMany processive, ring-ATPase motor proteins rely on substrate-dependent conformational changes to assist with the loading of client substrates into the central pore of the enzyme and subsequent translocation. Using the Escherichia coli Rho transcription terminator as a model hexameric helicase, we show that two distinct ligands--the antibiotic bicyclomycin and pyrimidine-rich nucleic acids--respectively repress or promote the transition of Rho from an open RNA-loading configuration to a closed-ring active helicase. Our findings explain several mechanisms by which Rho activity is controlled and provide a general illustration of how intrinsic and extrinsic factors can regulate ring-type ATPase dynamics through diverse mechanisms. 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 Rhos 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.
关键词：antibiotic ; ATPase ; helicase ; motor protein ; transcription
