Fig. 2: Effects of THB on subunit association and initiator tRNA binding during translation initiation. | Nature Communications

Fig. 2: Effects of THB on subunit association and initiator tRNA binding during translation initiation.

From: Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria

Fig. 2

a Time course of 50S association to 30S pre-IC in the presence of all three IFs, with or without THB. Subunit association was monitored as an increase in Rayleigh light scattering at 365 nm. Solid lines are the fits of data to complex formation equation (Supplementary Eq. 9). b The bars represent the extent of bound f[3H]Met-tRNAfMet to the mRNA-programmed 70S ribosomes in the presence of all IFs after incubation at 37 °C for 15 min (n = 3). THB was either added after 70S IC formation (marked IM + THB) or was pre-incubated with 70S for 5 min before addition of mRNA, f[3H]Met-tRNAfMet and the IFs (marked 70S + THB). c Real-time fluorescence traces of BOP•Met-tRNAfMet binding to mRNA-programmed 70S ribosomes in the presence and (d) absence of IFs. THB was pre-incubated with the ribosomes before mixing with BOP•Met-tRNAfMet in a stopped-flow instrument. Data were fitted to complex formation equation (Supplementary Eq. 8) to estimate the effective rate of binding without (kb) and with THB (kb*). Experiments were conducted in triplicates and error bars indicate SEM.

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