The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation
- Uppsala University, Sweden
Figures
Figure 1
Kinetics of tRNA selection in the presence of viomycin.
(A) Time courses of [3H] GDP formation by Phe-tRNAPhe containing EF-Tu TCs reacting with 0.5 µM 70S ribosomes with fMet-tRNAfMet in the P site, displaying either a cognate UUC or near-cognate CUC codon in the A site, at the indicated viomycin concentrations. Solid lines represent fits of single exponential equations to the data. (B) of GTP hydrolysis by Phe-tRNAPhe containing TCs estimated from experiments such as those in (A). Solid lines represent either a fit of a constant value (UUC) or Equation 3 (CUC) to the data. (C) Time courses of f[3H] Met-Phe dipeptide formation for 1 µM Phe-tRNAPhe containing EF-Tu TCs reacting with 70S ribosomes with f[3H]Met-tRNAfMet in the P site, displaying either a cognate UUC or near-cognate CUC codon in the A site, at the indicated viomycin concentrations. Solid lines represent fits of single exponential equations to the data. (D) of dipeptide formation by Phe-tRNAPhe containing EF-Tu TCs estimated from experiments such as those in (C). Solid lines represent either a fit of a constant value (UUC) or Equation 3 (CUC) to the data. All error bars represent SEM.
Figure 2
Stabilization of ternary complex on the ribosome by viomycin on both cognate and near-cognate codons.
(A) Time courses of chase experiments were f[3H]Met-Phe dipeptide is formed after chasing of 5 µM containing EF-TuH84A TCs from 70S ribosomes with f[3H] Met-tRNAfMet in the P site, displaying a cognate UUC codon in the A site, by 0.5 µM containing EF-TuWT TCs at the indicated viomycin concentrations. Solid lines represent fits of single exponential equations to the data. (B) Mean times of f[3H] Met-Phe dipeptide formation reflecting the mean times of containing EF-TuH84A TC dissociation estimated from experiments such as those in (A). Error bars represent SEM. The solid line represents a linear fit to the data. (C) Time courses of chase experiments where f[3H]Met-Leu dipeptide is formed after chasing of 5 µM Phe-tRNAPheGAA containing EF-TuH84A TCs from 70S ribosomes with f[3H] Met-tRNAfMet in the P site, displaying a near-cognate CUC codon in the A site, by 0.5 µM containing EF-TuWT TCs at the indicated viomycin concentrations. Solid lines represent fits of single exponential equations to the data.
Figure 3
Kinetic model for viomycin action during tRNA selection.
TC binds to a ribosome with an empty A site with rate constant k1 to form a viomycin-insensitive initial binding complex where the codon·anticodon interaction is not yet established. From this state either the TC dissociates with rate constant q2 or the ribosome proceeds along the selection pathway, with rate constant k2 to codon anticodon contact. From this state the ribosome can either return to the initial binding state with rate constantq3or proceed to a viomycin-sensitive state, with rate constant k3, where the codon·anticodon interaction is monitored by the activated monitoring bases A1492 and A1493. In this state three events can take place; the ribosome can return to the previous state with rate constant q4, the ribosome can proceed with hydrolysis of EF-Tu bound GTP with rate constant k4 or viomycin can bind with rate constant kvio. After GTP hydrolysis viomycin-free ribosomes can either form a peptide bond with rate constant k5 or reject the tRNA in the A site with rate constant kF. Viomycin-bound ribosomes are unable to reject the tRNA present in the A site and therefore will always proceed with GTP hydrolysis and peptide bond formation regardless of the nature of the codon·anticodon interaction.
Figure 4
Correlation between initial selection accuracy and viomycin sensitivity for four codon∙anticodon pairs.
(A) Time courses of f[3H]Met-Phe dipeptide formation for 1 µM Phe-tRNAPheGAA containing TCs reacting with 70S ribosomes, displaying either a cognate UUC codon or one of the near-cognate codons CUC, AUC, UAC or UUAin the A site (the underlined base differs from the cognate codon), in the presence of 250 µM viomycin. Solid lines represent fits of single exponential equations to the data. (B) of dipeptide formation by Phe-tRNAPheGAA containing TCs reacting with 70S ribosomes, displaying the near-cognate codons CUC, AUC, UAC or UUAin the A site, at varying concentrations of viomycin. Solid lines represent fits of Equation 3 to the data. (C) The accuracy of initial selection for Phe-tRNAPhecontaining TCs reading the indicated codons plotted against the concentration of viomycin required to increase the efficiency of each near-cognate reaction to half that of the cognate reaction ( ). The dotted line is a linear regression of the data illustrating the correlation predicted by Equation 2. All error bars represent SEM.
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- https://doi.org/10.7554/eLife.46124.006
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The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation
eLife 8:e46124.
https://doi.org/10.7554/eLife.46124
