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 protein 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 two different plasmid vector systems, in both streptomycin-sensitive and streptomycin-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 conserved leucine residue (L56-->H), associated with the phenotype of sensitivity to mechanical vibration and hemizygous female lethality in Drosophila, creates a functionally inactive but structurally stable protein that is not assembled into ribosomes. The presence in the cell of the mutant, but not wild-type, rpsL greatly downregulates the level of a prominent polypeptide of approximately 50 kDa. These results indicate novel structure-function relationships in rps12 genes affecting translational function, ribosome assembly and drug sensitivity, and indicate a novel regulatory pathway that may influence ribosome biogenesis.
Toivonen
J M
JM
Institute of Medical Technology, Tampere, Finland.
Boocock
M R
MR
Jacobs
H T
HT
D002455:Cell Division; D003001:Cloning, Molecular; D018389:Codon, Nonsense / Q000378:metabolism; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D004352:Drug Resistance, Microbial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D024101:Mitochondrial Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009596:Nitrophenols / Q000378:metabolism; D010641:Phenotype; D014176:Protein Biosynthesis; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013307:Streptomycin / Q000494:pharmacology; D013997:Time Factors
D018389:Codon, Nonsense; D017931:DNA Primers; D029968:Escherichia coli Proteins; D024101:Mitochondrial Proteins; D009596:Nitrophenols; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C112310:mitochondrial ribosomal protein S12; D013307:Streptomycin