Fig. 5: Effects of amikacin on the kinetics of peptide release and ribosome recycling.
From: Molecular basis of the pleiotropic effects by the antibiotic amikacin on the ribosome
a Time courses of BOP-Met-Phe-Leu release from the P site of the ribosomes in pre-TC (0.1 μM) upon mixing with RF2 (1 μM) in the presence of various concentrations of AMK (0-1 μM). The near monophasic curves are fitted with double exponential function (solid lines) and the rates and amplitudes of the predominant fast phase (> 99%) were determined. The fraction inhibited was estimated from the fractional loss in fluorescence amplitude for a given AMK concentration considering the total amplitude of fluorescence transition (without AMK) as 1. b Fraction inhibition of RF2-mediated peptide release as the function of increasing concentrations of AMK. Solid line is the hyperbolic fit of data from which half-maximal inhibitory concentration (KI) of AMK for peptide release was estimated. c Time traces for Rayleigh light scattering upon splitting of post-TC ribosomes (0.5 μM) into subunits by the concerted action of RRF (20 μM) and EF-G (10 μM) in the presence of various concentrations of AMK (0–20 μM). The scattering traces were fitted with double exponential function and the rates and amplitudes of both the fast and slow phases were determined. d Fraction inhibition of RRF and EF-G-mediated ribosome splitting was estimated from the fractional loss of the amplitude of the fast phase considering amplitude of the entire transition without AMK as 1. The solid line represents the hyperbolic fit of the fraction inhibition plotted against AMK concentration from which the half-maximal concentration (KI) of AMK to inhibit ribosome recycling was estimated. Experiments were conducted in triplicates, data were fitted in Origin(Pro), Version 2016 (OriginLab Corp.), and error bars indicate the SEM of data.
