In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli.
The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fusA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42 degrees C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within fusA. The fusA100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fusA101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of beta-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo. These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.
Hou
Y
Y
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Lin
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YP
Sharer
J D
JD
March
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PE
D000483:Alleles; D000595:Amino Acid Sequence; D002455:Cell Division; D003001:Cloning, Molecular; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics; D005816:Genetic Complementation Test; D006358:Hot Temperature; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000096:biosynthesis
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D001616:beta-Galactosidase