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 i s a highly conserved, essential activity of all cells. The precise mov ement of one codon that occurs following peptide bond formation is reg ulated by elongation factor G (EF-G) in eubacteria or elongation facto r 2 (EF-2) in eukaryotes. To begin to understand molecular interaction s that regulate this process, a genetic selection was developed with t he aim of obtaining conditional-lethal alleles of the gene (fusA) that encodes EF-G in Escherichia coli. The genetic selection depends bn th e observation that resistant strains arose spontaneously in the presen ce of sublethal concentrations of the antibiotic kanamycin. Replica pl ating was performed to obtain mutant isolates from this collection tha t were restrictive for growth at 42 degrees C. Two tightly temperature -sensitive strains were characterized in detail and shown to harbor si ngle-site missense mutations within fusA. The fusA100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fusA101 allele enco ded a glutamine-to-proline alteration at position 495. Induction kinet ics of beta-galactosidase activity suggested that both mutations resul ted in slower elongation rates in vivo. These missense mutations were very near a small group of conserved amino acid residues (positions 48 3 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded t hat these sequences encode a specific domain that is essential for eff icient translocase function.