Format

Send to

Choose Destination
J Biochem Biophys Methods. 2000 Jan 3;42(1-2):1-14.

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

Author information

1
Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, The Netherlands.

Abstract

Elongation factor Tu is essential for binding and a correct delivery of aminoacyl-tRNA during protein biosynthesis. For a good characterization of its interaction with tRNA in terms of structure-function relationship, determinations of kinetic equilibrium parameters are of great value. We describe two 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 Kd value together with the corresponding dissociation and association rate constants from one single set of experiments. The other is a rapid method for screening relative affinities of mutant EF-Tus for tRNA. It is based on competition between EF-Tu species with and without a (His)6 extension for the same aminoacyl-tRNA and yields a relative Kd value. The method can be of general importance for the measuring of ligand affinities of all sorts of His-tagged proteins. Both methods are illustrated by their application in the analysis of mutant EF-Tus with changed interactions with tRNA and antibiotics. Raising the assay temperature from 4 to 37 degrees C causes a 30-fold increase of Kd for EF-Tu x GTP x Phe-tRNA complexes. The mutation K237E leads to rapid inactivation at the latter temperature. A parallel is found between the order of increasing Kd values for EF-Tus with mutation G316D, A375T and Q124K, respectively, and their order of increasing resistance to kirromycin.

PMID:
10647810
DOI:
10.1016/s0165-022x(99)00032-9
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center