Aminoglycoside ototoxicity has been related to a surprisingly large number of cellular structures and metabolic pathways. The finding that patients with mutations in mitochondrial rRNA are hypersusceptible to aminoglycoside-induced hearing loss has indicated a possible role for mitochondrial protein synthesis. To study the molecular interaction of aminoglycosides with eukaryotic ribosomes, we made use of the observation that the drug binding site is a distinct domain defined by the small subunit rRNA, and investigated drug susceptibility of bacterial hybrid ribosomes carrying various alleles of the eukaryotic decoding site. Compared to hybrid ribosomes with the A site of human cytosolic ribosomes, susceptibility of mitochondrial hybrid ribosomes to various aminoglycosides correlated with the relative cochleotoxicity of these drugs. Sequence alterations that correspond to the mitochondrial deafness mutations A1555G and C1494T increased drug-binding and rendered the ribosomal decoding site hypersusceptible to aminoglycoside-induced mistranslation and inhibition of protein synthesis. Our results provide experimental support for aminoglycoside-induced dysfunction of the mitochondrial ribosome. We propose a pathogenic mechanism in which interference of aminoglycosides with mitochondrial protein synthesis exacerbates the drugs' cochlear toxicity, playing a key role in sporadic dosedependent and genetically inherited, aminoglycoside-induced deafness.decoding ͉ mitochondria ͉ ribosomes ͉ toxicity ͉ translation

Despite the fact that important genetic diseases are caused by mutant mitochondrial ribosomes, the molecular mechanisms by which such ribosomes result in a clinical phenotype remain largely unknown. The absence of experimental models for mitochondrial diseases has also prevented the rational search for therapeutic interventions. Here, we report on the construction of bacterial hybrid ribosomes that contain various versions of the mitochondrial decoding region of ribosomal RNA. We show that the pathogenic mutations A1555G and C1494T decrease the accuracy of translation and render the ribosomal decoding site hypersusceptible to aminoglycoside antibiotics. This finding suggests misreading of the genetic code as an important molecular mechanism in disease pathogenesis.decoding ͉ mitochondria ͉ mutant rRNA ͉ ribosomes ͉ disease

Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S rRNA with its eukaryotic counterpart, resulting in bacterial hybrid ribosomes with a fully functional eukaryotic rRNA decoding site. Cell-free translation assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of higher eukaryotes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes, while the decoding sites of parasitic protozoa show distinctive drug susceptibility. Our findings suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function.

Peptide bond formation is the main catalytic function of the ribosome. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, aminoacyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.Ribosomes catalyze peptide bond formation between aminoacyltRNA (aa-tRNA) 4 bound to the A site of the ribosome and peptidyltRNA at the P site. The active site for peptide bond formation, the peptidyl transferase center, is located on the large (50 S) ribosomal subunit. High-resolution crystal structures of the 50 S subunit have revealed that the peptidyl transferase center is composed of RNA (23 S rRNA), with no protein within 15 Å of the active site (1, 2). This implies that peptide bond formation is catalyzed by RNA and, thus, the ribosome is a ribozyme.The peptide bond is formed as a result of nucleophilic attack by the ␣-amino group of aa-tRNA on the ester carbonyl group of peptidyltRNA. The first step is the deprotonation of the ␣-NH 3 ϩ group to create the nucleophilic NH 2 group. The pK a of the ␣-NH 3 ϩ group in aa-tRNA is estimated to be around 8, and it is likely that the proton is accepted by water (3). Subsequent nucleophilic attack of the ␣-NH 2 group on the electrophilic carbonyl group leads to the formation of the zwitterionic tetrahedral intermediate, which, by deprotonation, forms the negatively charged tetrahedral intermediate. The breakdown of the tetrahedral intermediate is initiated by donating a proton back to the leaving oxygen to form the products, i.e. P-site deacylated tRNA and A-site peptidyl-tRNA.In principle, the ribosome may catalyze the reaction by several mechanisms, such as proper positioning of the peptidyl and aminoacyl ends of the tRNAs in the active site in a conformation suitable for the spontaneous reaction, general base-acid catalysis during deprotonation and protonation, or electrostatic stabilization of the transition state(s) (4 -9). The pH dependence of peptidyl transfer reaction catalyzed by Escherichia coli ribosomes suggested the importance of an ionizing group of the ribosome with a pK a of 7.5, which could contribute to chemical...

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Aminoglycoside antibiotics that bind to the aminoacyl-tRNA site (A site) of the ribosome are composed of a common neamine core in which a glycopyranosyl ring is attached to position 4 of a 2-deoxystreptamine moiety. The core is further substituted by one (ribostamycin), two (neomycin and paromomycin), or three (lividomycin A) additional sugars attached to position 5 of the 2-deoxystreptamine. To study the role of rings III, IV, and V in aminoglycoside binding, we used isogenic Mycobacterium smegmatis ⌬rrnB mutants carrying homogeneous populations of mutant ribosomes with alterations in the 16S rRNA A site. MICs were determined to investigate drug-ribosome interactions, and the results were compared with that of the previously published crystal structure of paromomycin bound to the ribosomal A site. Our analysis demonstrates that the stacking interaction between ring I and G1491 is largely sequence independent, that rings III and IV each increase the strength of drug binding to the ribosome, that ring IV of the 6-NH 3 ؉ aminoglycosides compensates for loss of interactions between ring II and U1495 and between ring III and G1491, that the aminoglycosides rely on pseudo-base pairing between ring I and A1408 for binding independently of the number of sugar rings attached to the neamine core, that addition of ring V to the 6-OH 4,5-aminoglycoside paromomycin does not alter the mode of binding, and that alteration of the U1406 · U1495 wobble base pair to the Watson-Crick interaction pair 1406C-1495G yields ribosomal drug susceptibilities to 4,5-aminoglycosides comparable to those seen with the wild-type A site.

The 23S rRNA A2058G alteration mediates macrolide, lincosamide, and streptogramin B resistance in the bacterial domain and determines the selectivity of macrolide antibiotics for eubacterial ribosomes, as opposed to eukaryotic ribosomes. However, this mutation is associated with a disparate resistance phenotype: It confers high-level resistance to ketolides in mycobacteria but only marginally affects ketolide susceptibility in streptococci. We used site-directed mutagenesis of nucleotides within domain V of 23S rRNA to study the molecular basis for this disparity. We show that mutational alteration of the polymorphic 2057-2611 base pair from A-U to G-C in isogenic mutants of Mycobacterium smegmatis significantly affects susceptibility to ketolides but does not influence susceptibility to other macrolide antibiotics. In addition, we provide evidence that the 2057-2611 polymorphism determines the fitness cost of the 23S rRNA A2058G resistance mutation. Supported by structural analysis, our results indicate that polymorphic nucleotides mediate the disparate phenotype of genotypically identical resistance mutations and provide an explanation for the large species differences in the epidemiology of defined drug resistance mutations.antibiotics ͉ ketolides ͉ ribosomes ͉ structure ͉ conformation M any groups of clinically useful antibiotics prevent the synthesis of new proteins by interacting with the bacterial ribosome (1). A prominent region targeted by ribosomal drugs is located around the peptide bond formation site (the peptidyl transferase center) and the entrance of the nascent protein exit tunnel on the large (50S) ribosomal subunit. The upper region of the tunnel provides the binding site for macrolide, lincosamide, and streptogramin B (MLS B ) antibiotics (2-6). The primary inhibitory effect of the lincosamide drugs is to inhibit the formation of peptide bonds; the main inhibitory effect of macrolides is to block the passage of the newly synthesized peptide chain through the exit tunnel (2).Macrolides have a common core structure formed by a lactone ring of different sizes (7). Ketolides are the latest derivatives developed from the macrolide erythromycin to improve antimicrobial activity. They are equipped with 3-keto and 6-methoxy groups that improve acid stability and allow drug binding without evoking resistance mediated by inducible erm genes (reviewed in ref. 8). Additional features are a cyclic carbamate at C11͞C12 and an extended arm that provides additional interactions with domain II of 23S rRNA (4, 5, 9-11): an alkyl-aryl extension bound to the cyclic carbamate in the case of telithromycin and a quinolylallyl group tethered to the O-6 position in the case of ABT-773.Resistance to macrolides occurs predominantly by modification of the drug-binding site and͞or by drug efflux. Target modification may be the result of two different mechanisms: (i) modification in trans conferred by erm genes, which methylate the adenine of 23S rRNA position 2058 and (ii) modification in cis, including mutational altera...

Most of our understanding of ribosome function is based on experiments utilizing translational components from Escherichia coli. It is not clear to which extent the details of translation mechanisms derived from this single organism are true for all bacteria. Here we investigate translation factor-dependent reactions of initiation and elongation in a reconstituted translation system from a Gram-positive bacterium Mycobacterium smegmatis. This organism was chosen because mutations in rRNA have very different phenotypes in E. coli and M. smegmatis, and the docking site for translational GTPases, the L12 stalk, is extended in the ribosomes from M. smegmatis compared to E. coli. M. smegmatis genes coding for IF1, IF2, IF3, EF-G, and EF-Tu were identified by sequence alignments; the respective recombinant proteins were prepared and studied in a variety of biochemical and biophysical assays with M. smegmatis ribosomes. We found that the activities of initiation and elongation factors and the rates of elemental reactions of initiation and elongation of protein synthesis are remarkably similar with M. smegmatis and E. coli components. The data suggest a very high degree of conservation of basic translation mechanisms, probably due to coevolution of the ribosome components and translation factors. This work establishes the reconstituted translation system from individual purified M. smegmatis components as an alternative to that from E. coli to study the mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria.