Interaction of the isolated domain II/III of<i>Thermus thermophilus</i>elongation factor Tu with the nucleotide exchange factor EF-Ts

Marynen, Peter; Reiser, Christian O. A.; Schirmer, Norbert K.; Kiefhaber, Thomas; Ott, Günther; Grillenbeck, Norbert; Sprinzl, Mathias

doi:10.1093/nar/18.23.6889

Cited by 32 publications

(26 citation statements)

References 37 publications

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“…The prokaryotic fragment of EF-Tu, enclosing domain I1 and 111, binds EF-Ts in Thermus thermophilus [41]. This observation is more or less in line with ours, showing binding of EF-1P to the carboxy-terminal part of EF-la.…”

Section: Discussionsupporting

confidence: 90%

Identification of the sites in the eukaryotic elongation factor 1α involved in the binding of elongation factor 1β and aminoacyl‐tRNA

Damme¹,

Amons²,

Möller³

1992

European Journal of Biochemistry

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In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1P and aminoacyl tRNA to the a-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemiu. For this purpose the polypeptide chain of EF-la, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-la and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129 -461 and 69 -461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1 -36, that remained tightly bound. The ability of the mixture of the 37 + 43-kDa fragments, including this amino-terminal peptide 1 -36, to bind GDP or to facilitate aminoacyl tRNA binding to saltwashed ribosomes was severely reduced, compared to intact EF-la. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1P . A 30-kDa fragment, comprising the residues 1 -287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1P was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1P is induced through binding of this factor to the carboxy-terminal part of EF-la. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-la, especially in the region of its presumed effector loop is induced.The most abundant eukaryotic translational factor is elongation factor 1 (EF-I), which catalyzes, together with the translocation factor EF-2, the elongation cycle in the eukaryotic protein synthesis [I, 21. EF-1 contains four subunits (a, /J, y and 6). The elongation starts with the formation of a ternary complex between EF-la, aminoacyl tRNA and GTP. Subsequently, aminoacyl tRNA is attached to the mRNAribosome complex. This latter step requires hydrolysis of GTP [2]. The elongation rate is increased by two other subunits, EF-1P and EF-16, which both accelerate the exchange of GDP, bound to EF-la, for GTP [2, 31. EF-1P and EF-16 have been isolated in complex with EF-1y as heterodimers or heterotrimers (EF-Ifiy or EF-lPyG, respectively). Together with EF-1 a, these EF-IPy(6) complexes form high-moleclarmass aggregates, which have been described in many eukaryotic species [4 -81. Furthermore, these aggregates have been found associated with valyl tRNA synthetase [9, 101.

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Section: Discussionsupporting

confidence: 90%

Identification of the sites in the eukaryotic elongation factor 1α involved in the binding of elongation factor 1β and aminoacyl‐tRNA

Damme¹,

Amons²,

Möller³

1992

European Journal of Biochemistry

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“…On the other hand, it was demonstrated that a polypeptide consisting of domains I1 and 111 of EF-Tu interacts with EF-Ts (Peter et al, 1990a). We assume therefore that an interaction of domain I with EF-Ts is possible only when a complete polypeptide chain including domain I11 is present.…”

Section: Discussionmentioning

confidence: 92%

“…The preparation of EF-Tu"'"' was described in detail previously (Peter et al, 1990a). It was obtained by digestion of nucleotide-free 7: thermophilus EF-Tu with Staphylococcus ciureus V8 protease and purified by gel permeation chromatography.…”

Section: Purification Of Ef-tu Variantsmentioning

confidence: 99%

“…It is expected that the domains I1 and I11 in the case of EF-Tu are essential for interac-tions involved in polypeptide chain elongation, i.e. the interactions with aminoacyl-tRNA, the ribosome and the nucleotide exchange factor EF-Ts (Ott and Sprinzl, 1992;Peter et al, 1990a;Tubulekas and Hughes, 1993).…”

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confidence: 99%

“…The deletion of the whole domain I does not lead to a decrease in thermostability since 7: thevmophilus EF-Tu""" confers similar heat resistance as the native EF-Tu (Peter et al, 1990a). Although the thermal stability of the isolated domain I of 7: thevmophilus EF-Tu was not directly measured, it was suggested on the basis of the tertiary structure of 7: thermoplzilus EF-Tu .…”

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

Nock

Grillenbeck

Ahmadian

et al. 1995

European Journal of Biochemistry

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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.

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Elongation Factors in Protein Synthesis

Kraal

Bosch

Mesters

et al. 2007

Ciba Foundation Symposium 176 ‐ the GTPase Superfamily

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Interaction of the isolated domain II/III ofThermus thermophiluselongation factor Tu with the nucleotide exchange factor EF-Ts

Cited by 32 publications

References 37 publications

Identification of the sites in the eukaryotic elongation factor 1α involved in the binding of elongation factor 1β and aminoacyl‐tRNA

Identification of the sites in the eukaryotic elongation factor 1α involved in the binding of elongation factor 1β and aminoacyl‐tRNA

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

Elongation Factors in Protein Synthesis

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