Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation

Two truncated variants of elongation factor G from Thermus thermophilus wit h deletion of its domain IV have been constructed and the mutated genes wer e expressed in Escherichia coli, The truncated factors were produced in a s oluble form and retained a high thermostability. It was demonstrated that m utated factors possessed (1) a reduced affinity to the ribosomes with an un cleavable GTP analog and (2) a specific ribosome-dependent GTPase activity. At the same time, in contrast to the mild-type elongation factor G, they w ere incapable to promote translocation, The conclusions are drawn that (I) domain IV is not involved in the GTPase activity of elongation factor G, (2 ) it contributes to the binding of elongation factor G with the ribosome an d (3) is strictly required for translocation, These results suggest that do main IV might be directly involved in translocation and GTPase activity of the factor is not directly coupled with translocation, (C) 1999 Federation of European Biochemical Societies.