Functional and structural studies on a tryptic fragment of eucaryotic elongation factor Tu from rabbit reticulocytes.

Treatment of eucaryotic elongation factor Tu (eEF-Tu; Mr 53 000) with trypsin results in cleavage of the factor at at least two sites, one and probably both of which are located near the amino-terminal end of the polypeptide chain. The products after exposure of eEF-Tu to trypsin for 2 h is a single polypeptide of 43 000 daltons (eEF-Tut) and as yet unidentified polypeptides of Mr less than or equal to 5000. The presence of high glycerol concentrations of GDP in the reaction mixture markedly retards the rate of tryptic cleavage, while GTP has little effect. When eEF-Tu is bound to eucaryotic elongation factor Ts in an eEF-T complex, it is much more resistant to the action of trypsin. The loss of factor activity during tryptic digestion (as measured by its ability to bind aminoacyl-tRNA to 80S (ribosomes) is much slower than the rate of eEF-Tut formation, and 2-h digests containing only eEF-Tut are about 30% as active as the native enzyme. However, no ribosome-dependent activity is detectable after purification of eEF-Tut by ion-exchange chromatography, followed by gel filtration. Purified eEF-Tut binds guanine nucleotides, although with diminished activity compared with that of eEF-Tu. Amino-terminal sequence analyses of eEF-Tut reveal a striking sequence homology with the functionally related factor from Escherichia coli (EF-Tu). The first four residues of eEF-Tut, Gly-Ile-Thr-Ile, are identical with the first four residues of a 37 000-dalton tryptic fragment of E. coli EF-Tu, and other homologies are evident in the first twelve amino-terminal residues of the corresponding tryptic fragments.