Probing Translation with Small-Molecule Inhibitors

Abstract

Summary The translational apparatus of the bacterial cell remains one of the principal targets of antibiotics for the clinical treatment of infection worldwide. Since the introduction of specific translation inhibitors into clinical practise in the late 1940’s, intense efforts have been made to understand their precise mechanisms of action. Such research has often revealed significant and sometimes unexpected insights into many fundamental aspects of the translation mechanism. Central to progress in this area, high-resolution crystal structures of the bacterial ribosome identifying the sites of antibiotic binding are now available, which, together with recent developments in single-molecule and fast-kinetic approaches, provide an integrated view of the dynamic translation process. Assays employing these approaches and focusing on specific steps of the overall translation process are amenable for drug-screening. Such assays, coupled with structural studies, have the potential not only to accelerate the discovery of novel and effective antimicrobial agents, but also to refine our understanding of the translation mechanism, since antibiotics often stabilize specific functional states of the ribosome and allow distinct translation steps to be dissected in molecular detail.

Four translation initiation pathways employed by the leaderless mRNA in eukaryotes

“…Comparatively, puromycin, a well-known aminonucleoside antibiotic that causes premature peptide chain termination3436, inhibited the cI-Fluc mRNA translation even stronger than did for the Actin-Rluc mRNA (Fig. 2b).…”

“…Antibiotics that target ribosomes are also a powerful tool for studying translation mechanisms3435. Both harringtonine and T-2 toxin block elongation by inhibiting peptidyl transferase center of the ribosome36.…”

“…It was fairly predictable for a non-specific elongation inhibitor, since the Fluc coding region is ~1.8 times longer than the Rluc one. Similarly to harringtonine and T-2 toxin, puromycin binds the A-site of the large ribosome subunit3436. However, contrary to the above two drugs, it enters 60 S subunit irrespectively of whether the latter is involved in the 80 S formation or not (Fig.…”

Ribosomes in a Stacked Array

“…In E. coli, puromycin reactivity of the A/P hybrid state ribosomes was ϳ10-fold lower than that of the post-translocation state ones (21,37,38 consider the majority first. The k app (V85 slow ) was similar to but significantly larger than k app (Pre) ( Table 2).…”

“…For the stacked ribosomes to stall at the post-translocation state and are prevented from proceeding to the peptidyltransfer step, either the release of eEF-2 or the incorporation of aminoacyl-tRNA must be inhibited (21,43). In prokaryotes, posttranslocation state ribosomes with associated EF-G exhibit full puromycin reactivity, although entry of aminoacyl-tRNA is possible only after dissociation of EF-G (21,38). This logic reveals an explanation for the remaining PtR(V85): the leading ribosome had initially arrested at Ser-94 and the second ribosome stacked at Val-85, but the leading ribosome has resumed translation (and thus was no longer detected at the Ser-94 position) before the start of puromycin reaction (Fig.…”