title: Construction of an in vivo nonsense readthrough assay system and functional analysis of ribosomal proteins S12, S4, and S5 in Bacillus subtilis.
abstract: To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in a ram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.
authors.affiliation: National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Inaoka
authors.forename: K
authors.initials: K
authors.lastname: Kasai
authors.forename: K
authors.initials: K
authors.lastname: Ochi
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins; D018389:Codon, Nonsense / Q000032:analysis* / Q000235:genetics; D029968:Escherichia coli Proteins; D007763:Lac Operon; D056968:Lac Repressors; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011072:Poly U / Q000378:metabolism; D014176:Protein Biosynthesis; D012097:Repressor Proteins; D012269:Ribosomal Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D018389:Codon, Nonsense; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D012269:Ribosomal Proteins; C034212:ribosomal protein S12; C031798:ribosomal protein S4; C030831:ribosomal protein S5; D011072:Poly U