A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3☆

Abstract We have investigated how point mutations in the two ATP-binding motifs (G 463 PNGCGK 469 ST and G 701 PNGAGK 707 ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae . The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine 463 and 701 and lysine 469 and 707 were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine 463 and 701 was replaced with valine. The mutant EF-3s did not support cell growth in TEF3 -disrupted S. cerevisiae , when each lysine 469 and 707 and glycine 463 was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae .