Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu

The presence or absence of deacylated tRNA at the E site sharply influences the activation energy required for binding of a ternary complex to the ribosomal A site indicating the different conformations that the E-tRNA imparts on the ribosome. Here we address two questions: (i) whether or not peptidyltransferase—the essential catalytic activity of the large ribosomal subunit—also depends on the occupancy state of the E site and (ii) at what stage the E-tRNA is released during an elongation cycle. Kinetics of the puromycin reaction on various functional states of the ribosome indicate that the A-site substrate of the peptidyltransferase center, puromycin, requires the same activation energy for peptide-bond formation under all conditions tested. We further demonstrate that deacylated tRNA is released from the E site by binding a ternary complex aminoacyl-tRNA•EF-Tu•GDPNP to the A site. This observation indicates that the E-tRNA is released after the decoding step but before both GTP hydrolysis by EF-Tu and accommodation of the A-tRNA. Collectively these results reveal that the reciprocal linkage between the E and A sites affects the decoding center on the 30S subunit, but does not influence the rate of peptide-bond formation at the active center of the 50S subunit

Investigating the entire course of telithromycin binding to Escherichia coli ribosomes

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (KT = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl–aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (KT* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes

Effect of polyamines on the inhibition of peptidyltransferase by antibiotics: revisiting the mechanism of chloramphenicol action

Chloramphenicol is thought to interfere competitively with the binding of the aminoacyl-tRNA 3′-terminus to ribosomal A-site. However, noncompetitive or mixed-noncompetitive inhibition, often observed to be dependent on chloramphenicol concentration and ionic conditions, leaves some doubt about the precise mode of action. Here, we examine further the inhibition effect of chloramphenicol, using a model system derived from Escherichia coli in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes, under ionic conditions (6 mM Mg(2+), 100 mM NH(4)(+), 100 µM spermine) more closely resembling the physiological status. Kinetics reveal that chloramphenicol (I) reacts rapidly with AcPhe-tRNA·poly(U)·70S ribosomal complex (C) to form the encounter complex CI which is then isomerized slowly to a more tight complex, C*I. A similar inhibition pattern is observed, if complex C modified by a photoreactive analogue of spermine, reacts in buffer free of spermine. Spermine, either reversibly interacting with or covalently attached to ribosomes, enhances the peptidyltransferase activity and increases the chloramphenicol potency, without affecting the isomerization step. As indicated by photoaffinity labeling, the peptidyltransferase center at which chloramphenicol binds, is one of the preferred cross-linking sites for polyamines. This fact may explain the effect of spermine on chloramphenicol binding to ribosomes

Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity ▿

Ketolides represent the latest generation of macrolide antibiotics, displaying improved activities against some erythromycin-resistant strains, while maintaining their activity against erythromycin-susceptible ones. In this study, we present a new ketolide, K-1325, that carries an alkyl-aryl side chain at C-13 of the lactone ring. According to our genetic and biochemical studies, K-1325 binds within the nascent polypeptide exit tunnel, at a site previously described as the primary attachment site of all macrolide antibiotics. Compared with telithromycin, K-1325 displays enhanced antimicrobial activity against wild-type Escherichia coli strains, as well as against strains bearing the U2609C mutation in 23S rRNA. Chemical protection experiments showed that the alkyl-aryl side chain of K-1325 interacts specifically with helix 35 of 23S rRNA, a fact leading to an increased affinity of U2609C mutant ribosomes for the drug and rationalizing the enhanced effectiveness of this new ketolide