FUNCTIONAL SUBDOMAINS OF YEAST ELONGATION-FACTOR-3 - LOCALIZATION OF RIBOSOME-BINDING DOMAIN

Elongation factor 3 (EF-3) is an essential requirement of the fungi fo r translational elongation, EF-3 is an ATPase, and the hydrolytic acti vity is stimulated 2 orders of magnitude by yeast ribosomes. Limited t rypsinolysis of EF-3 results in the cleavage of a single peptide bond between residues 774 (Arg) and 775 (Gin), generating polypeptides of a pproximate molecular mass 90 and 30 kDa. The 90-kDa fragment is relati vely resistant to proteolysis and retains ribosome-independent ATPase activity. The 30-kDa fragment is further proteolyzed into smaller frag ments and retains the specificity for binding to yeast ribosomes. Both the intact EF-3 and the 30-kDa fragment are protected from proteolysi s by yeast ribosomes, EF-3 is NH2 terminally blocked, and so is the 90 -kDa fragment. The COOH terminally derived 30-kDa fragment contains gl utamine (residue 775) at the NH2-terminal end. A construct was designe d representing the COOH terminal domain of EF-3 (30-kDa fragment), sub cloned, and expressed as a glutathione S-transferase fusion in yeast. The glutathione S-transferase-30-kDa peptide remains stringently assoc iated with ribosomes. Isolated fusion peptide rebinds to yeast ribosom es with high affinity, Eased on these results, we propose that at leas t one of the ribosome-binding sites of EF-3 resides at the COOH-termin al end of the protein.