Elongation factor (EF) Tu Thr-25 is a key residue binding the essential magnesium complexed to nucleotide. We have characterized mutations at this position to the related Ser and to Ala, which abolishes the bond to Mg 2؉ , and a double mutation, H22Y/T25S. Nucleotide interaction was moderately destabilized in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S). Binding Phe-tRNA Phe to poly(U)⅐ribosome needed a higher magnesium concentration for the latter two mutants but was comparable at 10 mM MgCl 2 . Whereas EFTu(T25S) synthesized poly(Phe), as effectively as wild type, the rate was reduced to 50% for EF-Tu(H22Y/T25S) and was, surprisingly, still 10% for EF-Tu(T25A). In contrast, protection of Phe-tRNA Phe against spontaneous hydrolysis by the latter two mutants was very low. The intrinsic GTPase in EF-Tu(H22Y/T25S) and (T25A) was reduced, and the different responses to ribosomes and kirromycin suggest that stimulation by these two agents follows different mechanisms. Of the mutants, only EFTu(T25A) forms a more stable complex with EF-Ts than wild type. This implies that stabilization of the EFTu⅐EF-Ts complex is related to the inability to bind Mg 2؉ , rather than to a decreased nucleotide affinity. These results are discussed in the light of the threedimensional structure. They emphasize the importance of the Thr-25-Mg 2؉ bond, although its absence is compatible with protein synthesis and thus with an active overall conformation of EF-Tu.
Elongation factor (EF)1 Tu belongs to the superfamily of guanine nucleotide-binding proteins, factors involved in numerous cellular processes as carrier of information or biological components. Common characteristics in the family are the switching between active and inactive conformations depending on whether GTP or GDP is bound (1, 2), an intrinsic GTPase activity that can be stimulated several orders of magnitude by specific factors (GTPase-activating proteins), and the property that the GDP/GTP exchange can be enhanced by specific exchange factors (guanine nucleotide exchange factors) (reviewed in Refs. 4 and 5). EF-Tu acts as carrier of aminoacyltRNAs (aa-tRNAs) to the ribosome in the elongation phase of bacterial protein biosynthesis. It has a long history of functional and structural studies offering many insights into its functioning (6). Structures of the two basic states of EF-Tu (7-10) and of the complexes with its guanine nucleotide exchange factor EF-Ts (11, 12) and with its "load" aa-tRNA (13) are now available. These provide snapshots of the dynamics of the factor during its cycle, which involves large interdomain rearrangements. On the other hand, many functional questions, such as the mechanism of GTP hydrolysis, the EF-Tu cycle on the ribosome, and the structure-function relationships of many structural elements, still remain unanswered or are awaiting satisfactory answers. Domain 1 of EF-Tu (G domain) has the guanine nucleotidebinding protein conserved fold and contains the "fingerprint" conserved sequence motifs taking care of the interaction w...