INVESTIGATION OF FUNCTIONAL-ASPECTS OF THE N-TERMINAL REGION OF ELONGATION-FACTOR TU FROM ESCHERICHIA-COLI USING A PROTEIN ENGINEERING APPROACH

The function of the N-terminal region of elongation factor Tu is still unexplained, Until recently, it has not been visible in electron dens ity maps from xray crystallography studies, but the presence of severa l well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly, In t his study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate, The resulting four point mutants were expressed and purified using the pGEX system, The untagged products were charac terized with regard to guanine-nucleotide interaction, intrinsic GTPas e activity, and binding of aminoacyl-tRNA (aa-tRNA), The results show that Lys(9) is especially strongly involved in the association with gu anine nucleotides and the binding of aa-tRNA. Also Lys(4) plays a role in the association of GDP and GTP and is also of some importance in a a-tRNA binding, Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interfac e between domains 1 and 2 with Lys(9) being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommoda ting the acceptor end of aa-tRNA as well as delineating the structure of the effector region.