Bacterial‐Protein Synthesis

Abstract

Bacterial protoplasts, treated with fusidic acid, have been used to examine the modes of action of various other antibiotics in vivo. This system, in which ribosomal function is severely impeded, is particularly useful for studying the actions of drugs which do not inhibit protein synthesis potently in vim. One such drug is spectinomycin which is here shown to be an inhibitor of translocation ; similar results were obtained with lincomycin and celesticetin. Althiomycin, conversely, was found to inhibit the peptidyl transferase reaction. Supplementary evidence for these conclusions was obtained by examining the effects of the drugs upon the isolated peptidyl transferase reaction in vivo and by studying their effects upon polyribosome stability. We conclude that lincomycin and celesticetin inhibit translocation only on those ribosomes which bear nascent peptides not exceeding a critical, but short, chain length. Recently, we have described the effects upon protein synthesis resulting from the addition of fusidic acid to intact bacterial protoplasts [l, 21. We now wish to demonstrate how fusidic acid-treated protoplasts can be used as a novel system for studying the modes of action of other antibiotics against bacterial protein synthesis in vivo.Fusidic acid inhibits protein synthesis by sequestering elongation factor G (EF-G) and guanosine diphosphate (GDP) on ribosomes [3] and the primary consequence of this effect is to prevent the binding of aminoacyl-tRNA into trhe ribosomal A site [l]. This sequence of events can be rationalized by hypothesizing that a ribosome possesses a single guanosine triphosphatase site, located in or near the 50-5 moiety of the A site, which is utilised alternately by EF-G during translocation of peptidyl-tRNA from the A to the P site and by elongation factor Tu (EF-Tu) during the binding of aminoacyl-tRNA into the A site [4,5].Inhibition of binding into the ribosomal A sites causes peptidyl-tRNA to be held in the P sites from which the nascent peptides can be readily released by incubation with puromycin. Thus, immediately following the addition of fusidic acid to protoplasts, treatment with puromycin results in almost quantitative release of such peptides. Subsequently, however the proportion of nascent peptides which resist release by puromycin gradually increases to around one third Abbreviations. EF-G, elongation factor G; EF-Tu, elongation factor Tu. of the total, at which point a steady-state is reached in which the nascent peptides enter and leave both A and P sites. Thus a greatly-slowed ribosomal cycle is eventually established in the presence of fusidic acid and nascent peptides are held for considerable periods of time in both A and P sites [Z]. Escape of nascent peptides from P sites can be explained by postulating breakdown of ribosome -EF-G -GDP -fusidic acid complexes thereby permitting binding of aminoacyl-tRNA into the now-vacant A sites and subsequent transfer of peptide moieties. Since these peptides are then constrained to remain in the A sites for some time (at le...

Inhibitors of protein synthesis

“…Althiomycin also blocks peptide bond formation in the intact bacteria [360]. As a consequence of their interaction with the Bacteriocin DFra is a protein, (mol.wt.…”

High Rates of Protein Synthesis by Isolated Chloroplasts

“…Chloramphenicol at a saturating concentration (0.4 mM) inhibited incorporation 97% (Iw1-l3 ,UM); cycloheximide had no effect (data not shown). Lincomycin is a potent inhibitor of chloroplast ribosomes (1 1), acting shortly after initiation on ribosomes bearing a short nascent chain (7). At a saturating concentration, lincomycin inhibited total protein synthesis 72%, and incorporation into peptides of the thylakoid membrane 83% (data not shown).…”

“…Shoots were cut in the light (275 ,umol m-2 s-1), immediately immersed in ice water, then homogenized within 7 min.…”

Enzymatic synthesis of aminocyclitol moieties of aminoglycoside antibiotics from inositol by Streptomyces spp.: detection of glutamine-aminocyclitol aminotransferase and diaminocyclitol aminotransferase activities in a spectinomycin producer

“…Structures of diaminocyclitol derivatives, with the antibiotics of which they are a constituent in parentheses. INN, streptamine (SS-56C; 2-hydroxygentamicin); dINN, 2-deoxystreptamine (gentamicin, neomycin); eINN, 2-epistreptamine (spectinomycin); eINNMe 2 , actinamine (spectinomycin); dINN 3 Me, 3 Me, 1-keto-2-deoxy-3-methylaminodeoxyscyllo-inositol; dIONЈMe, 1-methylaminodeoxy-2-deoxy-3-keto-scyllo-inositol; dIGNЈMe-P, 1-methylaminodeoxy-2-deoxy-3-guanidinodeoxy-6-phospho-scylloinositol.…”