Effect of Fusidic Acid on the Kinetics of Molecular Motions During EF-G-Induced Translocation on the Ribosome

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.

Figure 1

Fluorescence observables and translocation time courses recorded in in the absence (no antibiotic) or presence of fusidic acid (FA). Schematics of the translocation reaction highlight the positions of the fluorescent reporters. Assignment of fluorescence and FRET changes to distinct translocation steps of taken from. (a) FRET between L12-labeled with Alexa 488 and EF-G-labeled with QSY9 used to monitor EF-G binding to and dissociation from the ribosome. (b) Fluorescence change of Alexa 488 attached to S13 reflecting EF-G binding and further conformational rearrangements. (c) FRET between ribosomal proteins S6 and L9 labeled with Alexa 488 and Alexa 568, respectively, showing SSU body domain rotation relative to the LSU. (d) FRET between L33 labeled with Alexa 488 and S13 labeled with Atto540Q reporting on the SSU head domain swiveling. (e) Fluorescence change of fluorescein-labeled tRNA^fMet used to assess the tRNA movement from the P to the E site. (f) FRET between fluorescein attached to tRNA^fMet and Atto540Q on S13, which reports on the movement of the P-site tRNA to the E and E′ site and its dissociation from the ribosome.

Figure 2

Global analysis of translocation in the presence of FA. (a) Schematic of the 5-steps minimum model used for fitting. In step 1 (rate constants k₁ and k_-1), PRE complex binds EF-G (G) to form the PRE–EF-G complex which then undergoes several rearrangements leading to translocation and formation of the POST complex. Translocation intermediates, or chimeric (CHI) states, are adopted from previous structural work, ensemble kinetics and single molecule FRET experiments (reviewed in ref. 10). In step 2 (k₂), EF-G hydrolyzes GTP, engages in translocation, and uncouples SSU head and body movements, resulting in formation of an intermediate CHI1. This allows for the ribosome unlocking in step 3 (k₃), forming state CHI2. The following states CHI3 and CHI4 entail rapid Pi release from EF-G and stepwise tRNA translocation from the A to P and P to E sites. CHI3 and CHI4 have been characterized by cryo-EM and smFRET methods using specifically stalled complexes, but are not accumulating during unperturbed translocation and are therefore not observed as independent steps in ensemble kinetics; here formation of intermediated CHI2 to CHI4 is grouped into a single kinetic step. In step 4 (k₄), the tRNA that has been displaced from the P to the E site moves further through the ribosome via at least one distinct intermediate state (CHI5), and finally dissociates from the ribosome (k₅). Because EF-G does not dissociate in the presence of FA, the final state of the complex is POST–EF-G. (b) Translocation time-courses in the presence of FA (black) and the respective fits from numerical integration analysis (red). (c) Goodness of the fit as evaluated by KinTek Explorer FitSpace analysis. Dashed red lines reflect the lower and upper boundaries as calculated for a χ² threshold of 1.025 which reflects an estimate of the 95% confidence intervals of the elemental rates (see Manufacturer’s manual and ref. 35).

Figure 3

Intrinsic Fluorescence Intensities (IFI) values of the translocation intermediates. IFIs of intermediates during unperturbed translocation with EF-G–GTP (black; ref. 12) compared to IFIs obtained in the presence of FA (light-blue; this work).

Figure 4

Schematic of kinetic trapping of translocation by FA. The elemental rate constants of unperturbed translocation (black) compared to FA-impaired translocation (light blue). Step①, EF-G binding. Step②, EF-G engagement and uncoupling of the movements of the SSU head and body domains. Step③, ribosome unlocking, which allows for rapid synchronized tRNA movement from the A to P and P to E sites and Pi release from EF-G. Step④, movement of the E-site tRNA to the E′ state. Step⑤, dissociation of EF-G and tRNA leading to formation of the POST complex. The degree of the SSU head domain rotation is indicated by different shades of green, with dark green in the CHI1 state representing the maximum rotation. The degree of SSU body rotation relative to the LSU is shown in different shades of blue, with dark blue in the PRE–EF-G state displaying the maximum degree of body rotation.