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.

Figure 1.

Sequential strategy for the generation of a plasmid-rRNA exchange system. From top: Following deletion of chromosomal rrnA, a complementation vector pMIH-rrnB^2058G carrying a functional rrn operon was introduced to the chromosomal attB site. Subsequent deletion of rrnB resulted in M. smegmatis ΔrrnA ΔrrnB attB::pMIH-rrnB^2058G, in which ribosomal RNA is exclusively transcribed from the plasmid. From there, a plasmid-rRNA exchange system was established by replacing pMIH-rrnB^2058G with pMIG-rrnB-sacB. Transformation with hybrid rRNA genes pMIH-rrnB^hybrid and selection on sucrose resulted in M. smegmatis ΔrrnA ΔrrnB attB::pMIH-rrnB^hybrid with homogenous populations of hybrid ribosomes.

Figure 2.

16S rRNA sequence within helix 44 of M. smegmatis wild-type and hybrid ribosomes after transplanting the A-site rRNA of eukaryotic ribosomes. (A) Mycobacterium smegmatis. (B) Human–bacterial hybrid ribosomes. (C) Hybrid ribosomal RNA containing the decoding-site rRNA of the protozoan Leishmania, which is also identical to Trypanosoma; and (D) Blastocrithidia. Base substitutions rendering the bacterial 16S rRNA eukaryotic are depicted in blue; the transplanted region is boxed. rRNA residues are numbered according to the nucleotide numbering used in E. coli 16S rRNA.

Figure 3.

Kanamycin-induced inhibition of polypeptide synthesis using (UUU)₁₂-directed phenylalanine incorporation. (A) Homo sapiens cytosolic hybrid ribosomes (closed circles) versus M. smegmatis wild-type ribosomes (open circles). (B) Leishmania (closed squares) versus Blastocrithidia (open squares) hybrid ribosomes. The relative amount of [¹⁴C]-phenylalanine incorporated by 5 pmol of purified 70S ribosomes after 60 min. incubation in the presence of varying concentrations of kanamycin A is shown. SEs are indicated. The corresponding IC₅₀ values for kanamycin A and selected aminoglycoside antibiotics are given in Table 2.

Figure 4.

Paromomycin-induced inhibition of protein synthesis measured as luciferase activity in cell-free translation assays of firefly luciferase mRNA. Rabbit reticulocyte (closed triangles) versus human-bacterial hybrid (closed circles) and wild-type M. smegmatis (open circles) ribosomes; error bars represent the SEM (n = 3). The corresponding IC₅₀ values for paromomycin and the aminoglycosides tested are given in Table 3.