Abstract
The translocation step of protein synthesis entails binding and dissociation of elongation factor G (EF-G), movements of the two tRNA molecules, and motions of the ribosomal subunits. The translocation step is targeted by many antibiotics. Fusidic acid (FA), an antibiotic that blocks EF-G on the ribosome, may also interfere with some of the ribosome rearrangements, but the exact timing of inhibition remains unclear. To follow in real-time the dynamics of the ribosome–tRNA–EF-G complex, we have developed a fluorescence toolbox which allows us to monitor the key molecular motions during translocation. Here we employed six different fluorescence observables to investigate how FA affects translocation kinetics. We found that FA binds to an early translocation intermediate, but its kinetic effect on tRNA movement is small. FA does not affect the synchronous forward (counterclockwise) movements of the head and body domains of the small ribosomal subunit, but exerts a strong effect on the rates of late translocation events, i.e. backward (clockwise) swiveling of the head domain and the transit of deacylated tRNA through the E′ site, in addition to blocking EF-G dissociation. The use of ensemble kinetics and numerical integration unraveled how the antibiotic targets molecular motions within the ribosome-EF-G complex.
Highlights
IntroductionCryo-EM and X-ray might capture somewhat different states, these structures can be directly compared to the intermediates identified in kinetic studies[12, 21]
Max Planck Institute for Biophysical Chemistry, Department of Physical Biochemistry, Am Fassberg 11, 37077, Göttingen, Germany
Single-molecule fluorescence resonance energy transfer experiments indicated that Fusidic acid (FA) blocks elongation factor G (EF-G) in a late state after tRNA translocation, prior to the dissociation of the E-site tRNA from the ribosome
Summary
Cryo-EM and X-ray might capture somewhat different states, these structures can be directly compared to the intermediates identified in kinetic studies[12, 21] It is not clear when exactly FA binds to the ribosome–EF-G complex, how it affects the kinetics of elemental steps and the overall translocation trajectory, and how the rotation and swiveling of the body and head, respectively, respond to the FA binding. The antibiotic blocks the dissociation of tRNA from the E′ site, the completion of the backward swiveling of the SSU, and the release of EF-G These results provide a comprehensive picture of the mechanism of FA action and reconcile previous results of ensemble and smFRET kinetics and the structural work
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