SITE-DIRECTED MUTAGENESIS OF THERMUS-THERMOPHILUS EF-TU - THE SUBSTITUTION OF THREONINE-62 BY SERINE OR ALANINE

The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directe d mutagenesis by alanine and serine in the elongation factor Tu from T hermus thermophilus. The altered proteins were overproduced in Escheri chia coli, purified and characterized, The EF-Tu T62S variant had simi lar 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 po lypeptide synthesis and has only very low intrinsic and ribosome-induc ed 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 Mg2+ io n, gamma-phosphate of GTP and a water molecule, which is presumably in volved in the GTP hydrolysis.