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 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.
Vorstenbosch
E L
EL
Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, The Netherlands.
Potapov
A P
AP
de Graaf
J M
JM
Kraal
B
B
D001667:Binding, Competitive; D001671:Biochemistry / Q000379:methods*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine; D008956:Models, Chemical; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism
D010455:Peptides; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; C033223:polyhistidine; D006639:Histidine; D006160:Guanosine Triphosphate; D012259:Ribonuclease, Pancreatic; D010444:Peptide Elongation Factor Tu