Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Duisterwinkel, F. J.; Kraal, Barend; Graaf, J.Martien de; Talens, Anneke; Bosch, L.; Swart, G. M. W.; Parmeggiani, Andrea; Cour, T. F. M. La; Nyborg, Jens; Clark, Brian F. C.

doi:10.1002/j.1460-2075.1984.tb01770.x

Cited by 43 publications

(14 citation statements)

References 34 publications

(19 reference statements)

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“…Resistance of EF-Tu towards the antibiotic is induced by the point substitution of Ala375, a residue situated in the cleft between the N-terminal and the C-terminal domain, that is in an environment spatially at some distance from Lys357. Long-range effects cannot be excluded in this case [33]. It is known that interaction of kirromycin and EF-Ts is mutually exclusive [3]; the same holds for the interaction of GDP and EF-Ts with EF-Tu [l].…”

Section: The Gtpase Activity Of the G Domain As Afunction Of'the P Hmentioning

confidence: 99%

Structure‐function relationships of elongation factor Tu

Jensen

Cool

Mortensen

et al. 1989

European Journal of Biochemistry

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The guanine‐nucleotide‐binding domain (G domain) of elongation factor Tu(EF‐Tu) consisting of 203 amino acid residues, corresponding to the N‐terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141–3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF‐Tu towards thermal denaturation (t50%= 41.3°C vs. 46°C, respectively). Unlike EF‐Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF‐Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH4+, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF‐Tu. K+ and Na+ are the most active followed by NH4+ and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3–5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7–8, similar to that of EF‐Tu. The 70‐S ribosome is the only EF‐Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF‐Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF‐Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain GTP complex and aa‐tRNA. Our results indicate that the isolated G domain of EF‐Tu is a suitable model for studying the basic properties of the guanine nucleotide interaction and the catalytic activity of a guanine‐nucleotide‐binding protein.

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Section: The Gtpase Activity Of the G Domain As Afunction Of'the P Hmentioning

confidence: 99%

Structure‐function relationships of elongation factor Tu

Jensen

Cool

Mortensen

et al. 1989

European Journal of Biochemistry

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“…5B) was mutated to Cys. This residue, which corresponds to the Gly222-to-Asp mutation site in the original B 0 mutant of EF-Tu (18), is immediately adjacent to the F643R suppressor site. Second, Asn710 was mutated to cysteine.…”

mentioning

confidence: 99%

rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit

Shin

Kim

Acker

et al. 2009

Molecular and Cellular Biology

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The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.Four universally conserved GTPases interact with the ribosome to coordinate the initiation, elongation, and termination of protein synthesis. Initiation factor 2/eukaryotic translation initiation factor 5B (IF2/eIF5B) initially binds to the small ribosomal subunit and promotes subunit joining during translation initiation (5,30,36). The factor EF-Tu in bacteria or eEF1A in eukaryotes delivers aminoacyl-tRNAs to the A site of the ribosome, while the bacterial factor EF-G, or eEF2 in eukaryotes, promotes translocation of peptidyl-tRNA from the A to the P site during translation elongation (reviewed in references 1, 37, and 38). Finally, the factor RF3 (or eRF3 in eukaryotes) functions with the stop codon recognition factors to promote translation termination (25). As expected, the conserved GTP binding (G) domains of these factors contain the sequence motifs that are hallmarks of all G proteins. In addition to the Walker A-box GXXXGK(T/S) motif (G-1), which interacts with ␣ and ␤ phosphates of GTP, and the G-4 motif NKXD, which specifies guanine nucleotide recognition, the G-3 motif DXXG within the mobile switch II element is also conserved (48). Finally, X-ray structure analyses revealed that in addition to the G domain, the four translation GTPases share a conserved ␤-barrel domain II (28, 40). Binding of GTP versus GDP to EF-Tu altered the position of the switch II element and induced a significant rearrangement of domain II relative to the G domain (9, 26). Likewise, GTP binding to eIF5B induced movement of switch II and a rotation-like movement of domain II (39). As these gross structural rearrangements alter the aminoacyl-tRNA and/or ribosome binding affinities of the factors, it can be proposed that GTP switches govern the interaction of the translational GTPases with the ribosome.Cryo-electron microscopy (cryo-EM) studies have revealed the binding sites for EF-Tu, EF-G, IF2, and RF3 on the ribosome (2,3,20,27,33,47,49,55). All four factors were found to bind in the ribosomal entry site, the cleft on th...

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“…Apparently, a major site of interaction with aa-tRNA is on domain I, rather distant from Ala-375, It is conceivable, however, that other sites of interaction exist and that replacement of Ala-375 might cause a direct steric hindrance at these sites. Alternatively, it has been suggested earlier [6] that a mutation so close to the interface of domains I and II, could influence their relative positions, preventing optimal aa-tRNA binding. Since ternary complex formation presumably imposes a conformational change on EFTu -GTP, it is conceivable and in agreement with the lower binding constants, that mutant EFTu .GTP needs more energy to undergo such a conformational change than its wild-type counterpart.…”

Section: Discussionmentioning

confidence: 99%

“…EF-Tu from two of these mutant strains has its alanine residue at position 375 replaced by another amino acid, i.e., threonine (EF-Tu(Ar) from strain LBE2045) or valine (EFTu(A) from strain D2216) [5,6]. Since Ala-375 is located in a region which is probably involved in the control of various conformational states of EFTu [6], it was of considerable interest to study the consequences of such a substitution for the function of EF-Tu. Previously the GTPase activity and + Present address: Pharmaceutical R&D Laboratories, Organon International, PO Box 20,534O BH Oss, The…”

Section: Introduction Elongationmentioning

confidence: 99%

Mutant species of EF‐Tu, altered at position 375, exhibit a reduced affinity for aminoacylated transfer‐RNAs

1985

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The interaction between EF-Tu GTP and aminoacyl-tRNA is shown to be influenced by mutations at site 375 of this three-domain protein. Site 375 is located in domain II near the interface with domain I [(1984) EMBO J. 3. 113-1201. Replacement of the alanine at this site by a threonine or valine residue results in lower binding constants with Phe-tRNA and Tyr-tRNA. as was evaluated by the hydrolysis protection technique. The data are discussed in the light of what is known about the three-dimensional structure of the protein and its interaction sites with aminoacyl-tRNA.

show abstract

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Cited by 43 publications

References 34 publications

Structure‐function relationships of elongation factor Tu

Structure‐function relationships of elongation factor Tu

rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit

Mutant species of EF‐Tu, altered at position 375, exhibit a reduced affinity for aminoacylated transfer‐RNAs

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