R. Kambampati et K. Chakraburtty, FUNCTIONAL SUBDOMAINS OF YEAST ELONGATION-FACTOR-3 - LOCALIZATION OF RIBOSOME-BINDING DOMAIN, The Journal of biological chemistry, 272(10), 1997, pp. 6377-6381
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.