PROPERTIES OF ISOLATED DOMAINS OF THE ELONGATION-FACTOR TU FROM THERMUS-THERMOPHILUS HB8

The relative contributions of the three domains of elongation factor Tu (EF-Tu) to the factor's function and thermal stability were established by dissecting the domains apart with recombination techniques. Domain I (EF-Tu(I)), domains I/II (EF-Tu(I/II)) and domain III (EF-Tu(III)) of the EF-Tu from Thermus thermophilus HB8 comprising the amino acids 1-211, 1-312 and 317-405, respectively, were overproduced in Escherichia coli and purified. A polypeptide consisting of domain II and III (EF-Tu(II/III)) was prepared by limited proteolysis of native EF-Tu with V8 protease from Staphylococcus aureus [Peter, M. E., Reiser, C. O. A., Schirmer, N. K., Kiefhaber, T., Ott, G., Grillenbeck, N. W. & Sprinzl, M. (1990) Nucleic Acids Res. 18, 6889-6893]. As determined by circular dichroism spectrometry, the isolated domains have the secondary structure elements found in the native EF-Tu. GTP and GDP binding as well as GTPase activity are maintained by the EF-Tu(I) and EF-Tu(I/II); however, the rate of GDP dissociation from EF-Tu(I) . GDP and EF-Tu(I/II) . GDP complex is increased as compared to native EF-Tu . GDP, reflecting a constraint imposed by domain III on the ability to release the nucleotide from its binding pocket located in domain I. A weak interaction of Tyr-tRNA(Tyr) with the EF-TuI . GTP suggests that domain I provides a part of the structure interacting with aminoacyl-tRNA. The domain III is capable of regulating the rate of GTPase in EF-Tu, since the polypeptide consisting only of domains I/II has a 39-fold higher intrinsic GTPase compared to the native EF-Tu. No in vitro poly(U)-dependent poly(Phe) synthesis was detectable with a mixture of equimolar amounts of domains I/II and domain III, demonstrating the necessity of covalent linkage between the domains of EF-Tu for polypeptide synthesis. In contrast to native EF-Tu and EF-Tu(II/III), EF-Tu(I) and, to a lesser extent the polypeptide consisting of domains I/II, are unstable at elevated temperatures. This indicates that domains II/III strongly contribute to the thermal stability of this T. thermophilus EF-Tu. Deletion of amino acid residues 181-190 from domain I of T. thermophilus EF-Tu decreases the thermostability to that of EF-Tu from E. coli, which does not have these residues. Interdomain interactions must be important for the stabilisation of the structure of domain I, since isolated T. thermophilus EF-Tu(I) is thermolabile despite the presence of the 181-190 loop. Nucleotide exchange factor Ts (EF-Ts), which promotes the dissociation of the nucleotide from native EF-Tu, does not accelerate the nucleotide exchange from EF-Tu(I) . GDP and EF-Tu(I/II) . GDP polypeptides.