The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation.

Mikael Holm,Suparna Sanyal,Måns Ehrenberg,Chandra Sekhar Mandava

doi:10.7554/elife.46124

Abstract

Applying pre-steady state kinetics to an Escherichia-coli-based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.

Highlights

Viomycin is the first discovered member of the tuberactinomycin class of bacterial protein synthesis inhibiting antibiotics (Ehrlich et al, 1951; Finlay et al, 1951), commonly used to treat infections by Mycobacterium tuberculosis strains resistant to first-line drugs (WHO, 2010)
A reaction mixture containing varying concentrations of viomycin and Phe À tRNAPGhAeA in ternary complex (TC) with EF-TuÁGTP was rapidly mixed in a quench-flow instrument with initiated 70S ribosomes displaying either cognate (UUC) or nearcognate (CUC) codons in the A site
Based on the results presented above, we have constructed a kinetic model for how viomycin reduces the fidelity of mRNA decoding (Figure 3)

Summary

Introduction

Viomycin is the first discovered member of the tuberactinomycin class of bacterial protein synthesis inhibiting antibiotics (Ehrlich et al, 1951; Finlay et al, 1951), commonly used to treat infections by Mycobacterium tuberculosis strains resistant to first-line drugs (WHO, 2010) It is a cyclic pentapeptide, that is naturally synthesized by a non-ribosomal peptidyl transferase (Thomas et al, 2003). For fast and accurate protein synthesis, the ribosome must select for GTP hydrolysis those TCs which contain an AA-tRNA with a base triplet (anticodon) cognate to the base triplet on the mRNA (codon) displayed in the ribosomal A site. Those cognate AA-tRNAs will be selected for A-site accommodation and peptidyl transfer. The accuracy by which the ribosome discriminates against a given codon. anticodon mismatch is defined as the ratio between the kcat=KM values of the cognate and the non-cognate reaction (Fersht, 1998)

Methods

Results

Conclusion