Properties of Isolated Domains of the Elongation Factor Tu from Thermus Thermophilus HB8

Abstract

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 1 (EF-Tu'), domains 1/11 (EF-Tu'"') and domain 111 (EF-Tu"') 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 I1 and I11 (EF-Tu""") was prepared by limited proteolysis of native EF-Tu with V8 protease from Staphylococcus aureus [Peter, M. E., Reiser, C. 0. A,, Schirmer, N. K., Kiefhaber, T., Ott, G., Grillenbeck, N. W. & Sprinzl, M. (1990) Nucleic Acids Rex 18, 6889-68931. 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-Tul and EF-Tu""; however, the rate of GDP dissociation from EF-Tu' . GDP and EF-Tu"" . GDP complex is increased as compared to native EF-Tu . GDP, reflecting a constraint imposed by domain 111 on the ability to release the nucleotide from its binding pocket located in domain I. A weak interaction of Tyr-tRNATy' with the EF-Tu' . GTP suggests that domain I provides a part of the structure interacting with aminoacyl-tRNA. The domain 111 is capable of regulating the rate of GTPase in EF-Tu, since the polypeptide consisting only of domains 1/11 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 111, demonstrating the necessity of covalent linkage between the domains of EF-Tu for polypeptide synthesis. In contrast to native EF-Tu and EFTu""", EF-Tu' and, to a lesser extent the polypeptide consisting of domains ILI, are unstable at elevated temperatures. This indicates that domains II/III strongly contribute to the thermal stability of this 7: thermophilus EF-Tu. Deletion of amino acid residues 181 -190 from domain I of 7: thermophilus EF-Tu decreases the thermostability to that of EF-Tu from E. coli, which does not have these residues. lnterdomain interactions must be important for the stabilisation of the structure of domain I, since isolated 7: thermophilus EF-Tu' 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' . GDP and EF-Tu"" . GDP polypeptides.

Limited Proteolysis and Amino Acid Replacements in the Effector Region ofThermus thermophilusElongation Factor Tu

“…Since both amino acids are located i n domain IT, it is assumed that the ribosome-induced conformational change of EF-Tu and GTPase stimulation are mediated by domain 11. This proposal is also evident from the high GTPase activity of a truncated EF-Tu that contains only domains I and 11, which was proposed to mimic a ribosome-stimulated situation (Nock et al, 1995). However, as demonstrated in this work, the interaction of EF-Tu with the ribosome probably leads to structural changes in the mobile elements of the effector region to allow GTPase stimulation.…”

Properties of Truncated Forms of the Elongation Factor 1α from the Archaeon Sulfolobus solfataricus

“…Both Ss(GM)EF-I(x and Ss(G)EF-la bind guanine nucleotides but with a different behaviour compared to that reported for Ec(G)EF-Tu (Jensen et al, 1989), Tt(GM)EF-Tu and Tt(G)EF-Tu (Nock et al, 1995). In fact, while the deletions in the EF-Tu provoked a lower affinity for guanine nucleotides, both truncated SsEF-la forms showed an affinity for GDP, GTP or GuoPP[NH]P that, compared to intact SsEF-la, was approximately one order of magnitude higher (Table 1).…”

Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP

“…The amino acid sequence suggests that the GTP binding domain resides in the C-terminal part of McrB, starting around amino acid 200. In the case of EFTu, isolation of a functional GTP-binding domain has been carried out successfully (Parmeggiani et al, 1987;Jensen et al, 1989;Pingoud et al, 1989;Nock et al, 1995). In order to ®nd out whether it is possible to separate the DNA and GTP binding partial activities of McrB, we produced deletion mutants comprising the N-terminal (aa 1 to 190) and the C-terminal half (aa 189 to 465) as GSTfusion proteins.…”