Site-directed mutagenesis of Thermus thermophilus EF-Tu: the substitution of threonine-62 by serine or alanine

Abstract

The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu. In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity. The affinity of aminoacyl-tRNA to the EF-Tu T62A·GTP complex is almost 40 times lower as compared to the native EF-Tu·GTP. These observations are in agreement with the tertiary structure of EF-Tu·GTP, in which threonine-62 is interacting with the Mg 2+ ion, γ-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.