Putting Antibiotic Action Into Context

RESEARCH HIGHLIGHTS

Nature Reviews Microbiology | Published online 31 Oct 2016; doi:10.1038/nrmicro.2016.165

correlated with ribosome stalling. These data suggest that chloramphenicol and linezolid inhibit translation in a site-specific manner, and Douglas Carr/Alamy that inhibition efficiency depends on the amino acid context in the nascent chain. ANTIMICROBIALS In the absence of the antibiotic, expression of the chloramphenicol- resistance gene cmlAL is repressed because its ribosome-binding site is Putting antibiotic action sequestered in the mRNA secondary structure; in the presence of chloram- into context phenicol, ribosomes are arrested at a specific codon in the leader ORF Ribosomes are the site of protein stalled on various mRNAs. If chlor of the inducible resistance gene, synthesis and one of the main targets amphenicol and linezolid function which triggers a conformational of antimicrobials in the bacterial cell. as universal inhibitors, the ribosome change in the mRNA that releases the The ribosomal peptidyl transferase preincubated with those compounds ribosome-binding site and activates center (PTC), which resides in the should stall at the initiator codon of the expression of the resistance gene. large ribosomal subunit, catalyses any mRNA; however, they found that The authors showed that chloram- peptide-bond formation and peptide ribosomes were stalled at specific phenicol stalls translation of the release. PTC-targeting antibiotics, downstream sites, with varying cmlAL leader ORF when alanine is such as the natural compound efficiencies. They went on to analyse present at the penultimate position of chloramphenicol or the synthetic drug-induced translational arrest the peptide, which suggests that the inhibitor linezolid, bind to the A-site at selected codons and searched for site-specific arrest of ribosomes at the in the PTC, and it was suggested that specific sequence signatures among regulatory ORF of resistance genes is this interaction prevents the correct amino acids encoded within nine governed by sequence specificity of positioning of the amino-acylated codons preceding the arrest site chloramphenicol action. In addition, ends of tRNAs (aa-tRNA) and thus (positions −1 to −9 in the peptide), linezolid also arrested ribosomes at peptide-bond formation. As binding the arrest codons (the P-site; the same site, which suggests that of aa-tRNAs at the A-site is a key position 0 in the peptide) and the not only natural antibiotics but also step during protein synthesis, it was following codon (the A-site; position synthetic drugs can induce natural suggested that chloramphenicol +1). Interestingly, the arrest sites for antibiotic resistance genes. and linezolid function as universal both drugs showed a strong enrich- In summary, this study reveals that inhibitors of translation. However, ment for alanine and, to a lesser PTC-targeting ribosomal antibiotics the model that chloramphenicol and extent, for serine and threonine at function in a context-specific manner, linezolid indiscriminately inhibit the the −1 position. However, there was dependent on the penultimate amino formation of any peptide bond was no preference for a specific codon, acid in the nascent chain and the in conflict with several observations, which suggests that the penultimate amino acids in the P-site and A-site the efficiency of including the differential inhibition amino acid in the nascent chain of the PTC. These findings highlight chloramphenicol of translation of specific mRNA rather than the mRNA sequence or that insights into the mechanism of and linezolid templates and the induction of the tRNA structure is important for action of ribosomal antibiotics are chloramphenicol resistance genes. In drug action. Drug-induced ribosome crucial for the development of novel to inhibit this study, Marks et al. showed that stalling at the Leu5 codon in the hns and more effective antimicrobials to translation is chloramphenicol and linezolid pref- gene was among the strongest arrest tackle bacterial resistance. site-specific and erentially stall ribosomes at specific sites, and in vitro mutagenesis exper- Andrea Du Toit mRNA locations rather than function iments revealed that replacing the depends on ORIGINAL ARTICLE Marks, J. et al. Context- as global inhibitors of peptide-bond penultimate alanine residue at posi- specific inhibition of translation by ribosomal the amino acid formation. tion −1 with any other amino acid antibiotics targeting the peptidyl transferase center. Proc. Natl Acad. Sci. http://dx.doi. context in the The authors used toeprinting greatly reduced ribosome stalling. org/10.1073/pnas.1613055113 (2016) nascent chain analysis and ribosome-profiling Moreover, the authors showed FURTHER READING Wilson, D. L. Ribosome- experiments to determine the that the presence of glycine codons in targeting antibiotics and mechanisms of bacterial resistance. Nat. Rev. Microbiol. 12, 35–48 (2014) position at which ribosomes were either the P-site or A-site negatively