Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA.

Ove Wiborg,Carsten Andersen,Jens Nyborg,Charlotte R Knudsen,Brian F.C Clark

doi:10.1074/jbc.271.34.20406

Abstract

Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA.

Highlights

The mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay
Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis
The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay

Summary

EXPERIMENTAL PROCEDURES

Construction of Mutants—Site-directed mutagenesis was performed according to Taylor et al [28]. 0.67 ␮M [3H]Phe-tRNAPhe (specific activity 574 cpm/pmol) was incubated with EF-Tu1⁄7GTP (0.05–2 ␮M) in 50 ␮l of PP-6 buffer (60 mM Tris/Cl, pH 7.8, 30 mM KCl, 30 mM NH4Cl, 6 mM MgCl2) at 0 °C for 30 min. In 200 ␮l of PP-6 buffer 1 ␮M EF-Tu1⁄7GTP is incubated with 0.3 ␮M [3H]Phe-tRNAPhe (specific activity 574 cpm/pmol) at 0 °C for 30 min. The factor mix (in polymix buffer) contained 2 mM ATP, 20 mM phosphoenolpyruvate, 100 ␮M GTP, 0.1 mg/ml pyruvate kinase, 0.006 mg/ml myokinase, 14.6 ␮M [14C]Phe-tRNA (specific activity 31 Ci/mol), 0.2 mg/ml EF-Ts, 0.1 mg/ml EF-G, and 30 nM EF-Tu. The tRNA was charged before being added to the mixtures. The concentration was calculated from the absorption at A260 (1 A260 ϭ 23 pmol)

RESULTS

TABLE II Kinetic constants for the intrinsic GTPase activity

DISCUSSION