Mapping Escherichia coli Elongation Factor Tu Residues Involved in Binding of Aminoacyl-tRNA

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 aminoacyltRNA, 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.

Interaction of EF‐Tu with EF‐Ts: substitution of His‐118 in EF‐Tu destabilizes the EF‐Tu·EF‐Ts complex but does not prevent EF‐Ts from stimulating the release of EF‐Tu‐bound GDP

“…Nevertheless, the rate of poly(Phe) synthesis with the EF‐TuH118G mutant only reaches about 50% of that obtained with wtEF‐Tu suggesting that the mutation uH118G involves other basic aspects of poly(Phe) synthesis and not only the interaction with EF‐Ts. A considerably lower rate of poly(U)‐directed poly(Phe) synthesis upon EF‐Ts stimulation was also observed with two other uHis‐118 mutants: EF‐TuH118A and EF‐TuH118E [19]. Certainly, the lower efficiency of poly(Phe) synthesis is at least partially due to a strongly impaired binding of the EF‐TuH118 mutants to aa‐tRNA, an interaction shown to be deeply affected by the mutation or uH118 photooxidation [5, 6, 16, 20].…”

Genetic and molecular analysis of the tRNA-tufB operon of the myxobacterium Stigmatella aurantiaca

“…Elongation factor EF-Tu is a monomeric protein of -44 kDa and one of the most abundant proteins in E.coli (4). Its amino acid sequence (5) has been established, as well as the tertiary structure of first its GTP binding domain and recently a larger trypsintreated form lacking residues 45-58 (6, and for a review 7). EF-Tu is present in two forms in various bacterial species.…”

Recognition and selection of tRNA in translation

“…3). The intrinsic arginine residue (Arg58), that is located in the switch I region of EF-Tu in a position homologous to that of the catalytic arginine in G a proteins, is not essential for GTP hydrolysis [27], and an extensive mutagenesis search did not identify any other side chains important for GTP hydrolysis in Escherichia coli EF-Tu [27][28][29][30].…”