Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity.

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.

Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis.

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.

Mitochondrial deafness alleles confer misreading of the genetic code.

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.

Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria.

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.

Binding of neomycin-class aminoglycoside antibiotics to mutant ribosomes with alterations in the A site of 16S rRNA.

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 DeltarrnB mutants carrying homogeneous populations of mutant ribosomes with alterations in the 16S rRNA A site.

Essential mechanisms in the catalysis of peptide bond formation on the ribosome.

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.

23S rRNA base pair 2057-2611 determines ketolide susceptibility and fitness cost of the macrolide resistance mutation 2058A-->G.

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.