Carboxyl-terminal amino acid residues in elongation factor G essential for ribosome association and translocation.

The translocation of ribosomes on mRNA is carried out by cellular machinery that has been extremely well conserved across the entire spectrum of living species. This process requires elongation factor G (EF-G, or EF-2 in archaebacteria and eukaryotes), which is a member of the GTPase superfamily. Using genetic techniques, we have identified a series of mutated alleles of fusA (the Escherichia coli gene that encodes EF-G) that were unable to support protein synthesis in vivo. These alleles encode proteins with point mutations at codons 495 (a variant with a Q-to-P change at codon 495 [Q495P]), 502 (G502D), and 563 (G563D) and a nonsense mutation at codon 608. Biochemical analyses demonstrated that EF-G Q495P, G502D, and delta 608-703 were not disrupted in guanine nucleotide binding but were deficient in ribosome-dependent GTP hydrolysis and guanine nucleotide-dependent ribosome association. We propose that all of these mutations are present in a domain that is essential for ribosome association and that GTP hydrolysis was deficient as a secondary consequence of impaired binding to 70S ribosomes.