The effect of mutations in EF-Tu on its affinity for tRNA as measured by two novel and independent methods of general applicability

Elongation factor Tu is essential for binding and a correct delivery of ami noacyl-tRNA during protein biosynthesis. For a good characterization of its interaction with tRNA in terms of structure-function relationship, determi nations of kinetic equilibrium parameters are of great value. We describe t wo novel methods for that purpose. One method is based on EF-Tu protection of the tRNA 3' acceptor end against RNase A cleavage and yields the K-d val ue together with the corresponding dissociation and association rate consta nts from one single set of experiments. The other is a rapid method for scr eening relative affinities of mutant EF-Tus for tRNA. It is based on compet ition between EF-Tu species with and without a (His)(6) extension for the s ame aminoacyl-tRNA and yields a relative K-d value. The method can be of ge neral importance for the measuring of Ligand affinities of all seas of His- tagged proteins. Both methods are illustrated by their application in the a nalysis of mutant EF-Tus with changed interactions with tRNA and antibiotic s. Raising the assay temperature from 4 to 37 degrees C causes a 30-fold in crease of K-d for EF-Tu.GTP.Phe-tRNA complexes. The mutation K237E leads to rapid inactivation at the latter temperature. A parallel is found between the order of increasing K-d values for EF-Tus with mutation G316D, A375T an d Q124K, respectively, and their order of increasing resistance to kirromyc in. (C) 2000 Elsevier Science B.V. All rights reserved.