Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria

Abstract

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.

Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity

“…Thus, A1408 and G1491 facilitate the proper insertion of ring I carrying either an amino or hydroxyl group into the binding site, whereas G1408 and A1491 cause the binding pocket to be shallower and less stable, thus preventing proper insertion and binding of ring I (Recht et al 1999b;Lynch and Puglisi 2001a;Westhof, 2001, 2003). Consistent with these findings, a recent study demonstrated that the introduction of the eukaryotic decoding site into bacteria 16S rRNA conferred aminoglycoside resistance comparable to eukaryotic ribosomes (Hobbie et al 2007).…”

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

“…Ribosome concentrations of 70S were determined by absorption measurements on the basis of 23 pmol ribosomes per A 260 unit. Integrity and functional activity of purified 70S ribosomes was determined by analytical ultracentrifugation and by assessing their capacity to form initiation complexes, as described previously (29).…”

“…A reaction mixture containing M. smegmatis tRNA bulk , amino acids, S100 extract, energy mix, pyruvate kinase, and polyamines was preincubated with 30 M [ 14 Cell-Free Luciferase Translation Assays. Purified 70S hybrid ribosomes were used in a coupled transcription-translation reaction as described previously (29). The reaction mixture was incubated for 60 min at 37°C, stopped on ice, and luciferase assay substrate (Promega) was added.…”

Mitochondrial deafness alleles confer misreading of the genetic code

“…Ribosomes were isolated from bacterial cell pellets as described (36). For further fractionation, isolated ribosomes were resuspended in overlay buffer [20 mM Tris⅐HCl (pH 7.4), 60 mM NH4Cl, 5.25 mM MgCl2, 0.25 mM EDTA, 3 mM 2-mercaptoethanol, and 5% sucrose] loaded on a sucrose gradient (10 -40% sucrose in overlay buffer) and centrifuged in a Beckman Ti 15 rotor at 28,000 rpm for 18 h. Gradient fractions were collected by unloading the zonal rotor with 50% sucrose in overlay buffer.…”

“…70S ribosome concentrations were determined by absorption measurements on the basis of 23 pmol per A 260 unit. Integrity and functional activity of purified 70S ribosomes was determined by analytical ultracentrifugation and by assessing their capacity to form initiation complexes, as described (36).…”