Mutation Analysis of the Role in Ribosomal Translocation for Loops of Domain IV of the Elongation Factor G

Seven variants of Thermus thermophilus elongation factor G (EF-G) with mutations in loops of domain IV were constructed by PCR. Point mutations Arg504 Thr, Pro554 Thr, or Ile534 Asp did not affect the GTPase and translocational activities of EF-G. Similar results were obtained for mutants with tetra- or hexapeptide inserts in two loops located at the tip and two loops at the base of domain IV. Insertion of tetrapeptide Gly-Ser-Gly-Thr into loop 501–504 at the tip of domain IV dramatically reduced the activity of EF-G in poly(U)-directed polyphenylalanine synthesis on ribosomes, and halved its translocational activity. The intact conformation of loop Thr501-Gly-Gly-Arg504 was assumed to be essential for sterically perfect, efficient interaction of EF-G with the ribosome. The structural and biochemical data on the 30S subunit and EF-G were analyzed to specify the position of EF-G relative to the 30S and 50S ribosomal subunits.