Site‐Directed Mutagenesis of Thermus thermophilus Elongation Factor Tu

Abstract

His85 in Thermus thermophilus elongation factor Tu (EF‐Tu) was replaced by glutamine, leucine and glycine residues, leading to [H85Q]EF‐Tu, [H85L] EF‐Tu and [H85G]EF‐Tu, respectively. Asp81 was replaced by alanine leading to [D81A]EF‐Tu, and replacement of Arg300 provided [R300I]EF‐Tu. Glycine in position 85 of domain I induces a protease‐sensitive site in domain II and causes complete protein degradation in vivo. A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine. Degradation is probably due to disturbed interactions between the domains of EF‐Tu · GTP, inducing a protease‐sensitive cleavage site in domain II. [H85Q]EF‐TU, which can be effectively overproduced in Escherichia coli, is slower in poly(U)‐dependent poly(Phe) synthesis, has lower affinity to aminoacyl‐tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein. The GTPase of this protein variant is not efficiently stimulated by aminoacyl‐tRNA and ribosomes. The slow GTPase of [H85Q]EF‐Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)‐dependent ribosomal translation. Replacement of His85 in T. thermophilus EF‐Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl‐tRNA binding. [H85L]EF‐Tu is inactive in poly(U)‐dependent poly(Phe)‐synthesis. The rate of nucleotide dissociation is highest for [H85L]EF‐Tu, followed by [H85Q]EF‐Tu and native T. thermophilus EF‐Tu. Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF‐Tu domains, nucleotide binding and the efficiency and rate of GTPase activity.