Modelling in Escherichia coli of mutations in mitoribosomal protein S12: novel mutant phenotypes of rpsL

The rpsL gene of Escherichia coli encodes the highly conserved rps12 protei n of the ribosomal accuracy centre, We have used the E. coli gene to model the phenotypic effects of specific substitutions found in the mitochondrial gene for rps12. Variants created by in vitro mutagenesis were tested in tw o different plasmid vector systems, in both streptomycin-sensitive and stre ptomycin-resistant hosts. A substitution with respect to eubacterial rps12 (K87-->Q), found in all metazoan and fungal mitochondrial orthologues thus far studied, is associated with low-level resistance to streptomycin and a modest (15%) drop in translational elongation rate, but without significant effects on translational accuracy. An amino-acid replacement at a highly c onserved leucine residue (L56-->H), associated with the phenotypes of sensi tivity to mechanical vibration and hemizygous female lethality in Drosophil a, creates a functionally inactive but structurally stable protein that is not assembled into ribosomes. The presence in the cell of the mutant, but n ot wild-type, rpsL greatly downregulates the level of a prominent polypepti de of approximate to 50 kDa. These results indicate novel structure-functio n relationships in rps12 genes affecting translational function, ribosome a ssembly and drug sensitivity, and indicate a novel regulatory pathway that may influence ribosome biogenesis.