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Mathias Sprinzl - One of the best experts on this subject based on the ideXlab platform.

  • In vitro selected RNA molecules that bind to Elongation Factor Tu.
    Biochemistry, 1998
    Co-Authors: Veronika Hornung, Hans-peter Hofmann, Mathias Sprinzl
    Abstract:

    RNA molecules which bind to Elongation Factor Tu from T. thermophilus were isolated from a pool of ribooligonucleotides with a randomized sequence region. These RNAs interact with Elongation Factor Tu in both the GTP and the GDP form. A slight preference for the GTP form of the protein was observed. The isolated RNA aptamers compete with each other for a common binding site on Elongation Factor Tu. This binding site is different from the binding site for aminoacyl-tRNA or the binding site for Elongation Factor Ts and is located on domain II of Elongation Factor Tu. The selected RNAs do not bind to Elongation Factor G. The EF-Tu binding RNAs share a short consensus sequence, 5'-ACCGAAG-3', which was also found in the alpha-sarcin domain of T. thermophilus23S rRNA. The isolated RNAs have a hairpin structure with the 5'-ACCGAAG-3' sequence located in non-base-paired regions. Chemical probing and deletion experiments indicate that the consensus sequence is required for the interaction with Elongation Factor Tu.

  • Structure and importance of the dimerization domain in Elongation Factor Ts from Thermus thermophilus.
    Biochemistry, 1996
    Co-Authors: Youxing Jiang, Mathias Sprinzl, Steffen Nock, Martina Nesper, Paul B. Sigler
    Abstract:

    Elongation Factor Ts (EF-Ts) functions as a nucleotide-exchange Factor by binding Elongation Factor Tu (EF-Tu) and accelerating the GDP dissociation from EF-Tu; thus EF-Ts promotes the transition o...

  • Elongation Factor Ts from Thermus thermophilus-- overproduction in Escherichia coli, quaternary structure and interaction with Elongation Factor Tu.
    European journal of biochemistry, 1996
    Co-Authors: Jutta Blank, Steffen Nock, Roland Kreutzer, Mathias Sprinzl
    Abstract:

    The gene encoding the Elongation Factor Ts from Thermus thermophilus was sequenced, cloned and the protein overproduced in Escherichia coli. In comparison to the EF-Ts from E. coli with 282 amino acid residues, EF-Ts from T. thermophilus is considerably shorter, differing by 86 amino acids. EF-Ts from the thermophile is stable at high temperatures, which facilitates its separation from E. coli proteins. Purified T. thermophilus EF-Ts forms a homodimer with a disulfide bridge between the two cysteine residues at position 190. The modification of Cys190 by iodoacetamide affects neither the dimerization nor the ability of EF-Ts to facilitate the nucleotide exchange of Elongation Factor Tu. The disulfide bridge was detected only in purified EF-Ts, but not in protein extracts immediately after cell disruption. The physiological role of this disulfide bridge remains, therefore, unclear. Besides the quaternary (EF-Tu · EF-Ts)2 complex, a ternary EF-Tu · EF-Ts2 complex was detected by gel permeation chromatography and polyacrylamide gel electrophoresis. Trypsin cleavage after Lys48 or modification of Cys78 yield inactive EF-Ts, that does not bind to EF-Tu but is still capable of forming homodimers.

  • Minimalist aminoacylated RNAs as efficient substrates for Elongation Factor Tu.
    Biochemistry, 1994
    Co-Authors: Joelle Rudinger, Bernd Blechschmidt, Sofia Ribeiro, Mathias Sprinzl
    Abstract:

    We demonstrate here, using RNA variants derived from tRNAAsp, that the minimalist aminoacylated structure able to interact efficiently with Elongation Factor Tu comprises a 10 base-pair helix linked to the 3'-terminal NCCA sequence. Shorter structures can interact with the Elongation Factor, but with significantly decreased affinity. Conserved features in the aminoacyl acceptor branch of tRNAs, such as base pair G53-C61 and the T-loop architecture, could be replaced respectively by the inverted base pair C53-G61 and by unusual anticodon loop or tetraloop sequences. Variants of whole tRNAAsp or of the 12 base-pair aspartate minihelix, with enlarged 13 base-pair long aminoacyl acceptor branches, as in selenocysteine-inserting tRNAs that are not recognized by Elongation Factor Tu, keep their binding ability to this Factor. These functional results are well accounted for by the crystallographic structure of the Thermus thermophilus binary EF-Tu.GTP complex, which possesses a binding cleft accommodating the minimalist 10 base-pair domain of the tRNA aminoacyl acceptor branch.

  • Discrimination against misacylated tRNA by chloroplast Elongation Factor Tu
    European journal of biochemistry, 1994
    Co-Authors: Manfred Stanzel, Astrid Schön, Mathias Sprinzl
    Abstract:

    Chloroplast Elongation Factor Tu was purified from Pisum sativum and the binding properties of glutamylated chloroplast tRNAs were studied by gel-permeation chromatography. Whereas chloroplast Glu-tRNAGlu is efficiently bound by this Factor, the misacylated Glu-tRNAGin does not interact with chloroplast Elongation Factor Tu · GTP and is thus efficiently excluded from protein synthesis. Comparison with the behaviour of Escherichia coli Elongation Factor Tu · GTP shows that this Factor, which is not confronted with the in vivo misacylation phenomenon of organelles, binds both Glu-tRNAGlu and Glu-tRNAGln from chloroplasts with approximately equal efficiency.

Scott C Blanchard - One of the best experts on this subject based on the ideXlab platform.

Charlotte R. Knudsen - One of the best experts on this subject based on the ideXlab platform.

  • The role of Glu259 in Escherichia coli Elongation Factor Tu in ternary complex formation.
    Protein engineering, 1998
    Co-Authors: Gitte Nautrup Pedersen, Thomas Rattenborg, Charlotte R. Knudsen, Brian F.c. Clark
    Abstract:

    Determination of the crystal structure of the ternarycomplex formed between Elongation Factor Tu:GTP andaminoacylated tRNA revealed three regions of interactionbetween Elongation Factor Tu and tRNA. The structureindicates that the conserved glutamic acid at position 271in Thermus aquaticus EF-Tu could be involved in thebinding of the 39 CCA-Phe end of the aminoacylatedtRNA. Therefore, the corresponding residue, Glu259, ofEscherichia coli EF-Tu was mutated into alanine, asparticacid, glutamine and tyrosine, in order to substantiate thecrystallographic structural evidence and to obtain furtherknowledge of the importance of this residue. All of themutated proteins showed nucleotide binding propertiessimilar to the wild type. In addition the GTPase activitieswere similar to the wild type. The mutation of Glu259 toeither alanine or aspartic acidresulted in a reduced strengthof interaction with tRNA, while mutation to tyrosine abol-ished completely the interaction with tRNA. Finally, muta-tion to glutamine resulted in an Elongation Factor Tu variantbehaving like the wild type. In conclusion, the environmentaround the site binding the CCA-Phe end of the tRNA isvery restricted spatially and chemically so that only aresidue with almost the same size and chemical propertiesas glutamic acid fulfils the requirements with regard tosize, salt bridge-formation potential and maintenance ofthe backbone conformation at the 259 position.Keywords: Elongation Factor Tu/site-directed mutagenesis/structure–function relations/ternary complexIntroductionElongation Factor Tu (EF-Tu) is one of the components in theElongation cycle of prokaryotic protein biosynthesis. EF-Tu isa G-protein, which in its active GTP-bound conformationserves as the carrier of aminoacylated tRNA (aa-tRNA) tothe A-site of a mRNA-programmed ribosome. Upon codon–anticodon interaction GTP hydrolysis is triggered and EF-Tuis converted into its inactive GDP-form, which has a lowaffinity for both the ribosome and the aa-tRNA. Therefore itdissociates from the ribosome leaving the aa-tRNA on theribosome. Interaction with Elongation Factor, EF-Ts, reactivatesEF-Tu, as EF-Ts catalyses the exchange of GDP for GTP (fora review see Kjeldgaard et al., 1996).EF-Tu also plays a role in the proofreading mechanismensuring the incorporation of the correct amino acid intothe nascent polypeptide chain (Hopfield, 1974; Ninio, 1975;Thompson and Stone, 1977; Ruusala et al., 1982).The following structures of EF-Tu in different complexes

  • CONTRIBUTION OF ARG288 OF ESCHERICHIA COLI Elongation Factor TU TO TRANSLATIONAL FUNCTIONALITY
    European journal of biochemistry, 1997
    Co-Authors: Thomas Rattenborg, Brian F.c. Clark, Gitte Nautrup Pedersen, Charlotte R. Knudsen
    Abstract:

    The recently solved structure of the ternary complex formed between GTP-bound Elongation Factor Tu and aminoacylated tRNA reveals that the elements of aminoacyl-tRNA that interact with Elongation Factor Tu can be divided into three groups: the T stem; the 3'-end CCA-Phe; and the 5' end. The conserved residues Arg288, Lys89 and Asn90 are involved in the binding of the 5' end. In the active, GTP-bound form of the Elongation Factor, Arg288 and Asn90 are involved in the formation of a network of hydrogen bonds connecting the switch regions I and II of domain 1 with the rest of the molecule. This network is disrupted upon formation of the ternary complex. Arg288 was replaced by alanine, isoleucine, lysine or glutamic acid, and the resulting mutants have been subjected to an in vitro characterisation with the aim of clarifying the function of Arg288. Unexpectedly, the mutants behaved like the wild-type Factor with regard to the association and dissociation of guanine nucleotides, and the intrinsic GTPasc activities are unchanged. Furthermore, the mutants were as efficient as the wild-type Factor in carrying out protein synthesis in vitro in the presence of an excess of aminoacyl-tRNA. However, the mutants' abilities to bind aminoacyl-tRNA and protect the labile aminoacyl bond were impaired, especially where the charge had been reversed.

  • Mapping Escherichia coli Elongation Factor Tu Residues Involved in Binding of Aminoacyl-tRNA
    The Journal of biological chemistry, 1996
    Co-Authors: Ove Wiborg, Charlotte R. Knudsen, Brian F.c. Clark, Carsten Andersen, Jens Nyborg
    Abstract:

    Abstract Two residues of Escherichia coli Elongation Factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of Elongation Factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange Factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of Elongation Factor Tu in complex with aminoacyl-tRNA.

  • Mutation of the Conserved Gly83 and Gly94 in Escherichia Coli Elongation Factor Tu
    European Journal of Biochemistry, 1995
    Co-Authors: Inger V. H. Kjærsgård, Charlotte R. Knudsen, Ove Wiborg
    Abstract:

    Elongation Factor Tu from Escherichia coli cycles between an active conformation where GTP is bound, and an inactive conformation where GDP is bound. Between the two conformations, Elongation Factor Tu undergoes major structural changes. The aim of this work has been to reveal the role of two very well conserved glycine residues, Gly83 and Gly94, in the switch mechanism. Gly83 has been mutated alone or in combination with Gly94, both glycine residues being mutated to alanine. Enzymic characterisation of the two mutants have shown that they have an altered nucleotide affinity, a decrease in aminoacyl-tRNA affinity, an increase in intrinsic GTP hydrolysis, different behaviours in effector stimulation of the intrinsic GTPase activity, and that they are completely unable to sustain poly(Phe) synthesis in an in-vitro poly(U)-directed system. Our results indicates that particularly Gly83 is an important pivot point in Elongation Factor-Tu.

  • One-step purification of E. coli Elongation Factor Tu.
    Biochemistry international, 1992
    Co-Authors: Charlotte R. Knudsen, Brian F.c. Clark, B. Degn, Ove Wiborg
    Abstract:

    The tuf A gene, encoding the E. coli Elongation Factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one-step procedure. The construct was designed in order to make possible the release of authentic EF-Tu by cleaving the fusion protein with the protease Factor Xa.

Benjamin J Burnett - One of the best experts on this subject based on the ideXlab platform.

Norbert K. Schirmer - One of the best experts on this subject based on the ideXlab platform.

  • Crystals of intact Elongation Factor Tu fromThermus thermophilus diffracting to 1.45A˚resolution
    Journal of Crystal Growth, 1992
    Co-Authors: L.s. Reshetnikova, R. Hilgenfeld, Christian O. A. Reiser, Norbert K. Schirmer, H. Berchtold, R. Storm, Mathias Sprinzl
    Abstract:

    Intact Elongation Factor Tu fromThermus thermophilus HB8 was crystallized in the presence of guanosine-5'-diphosphate (GDP), guanosine-5'-(β,γ-imido)triphosphate (GPPNP), guanosine-5'-(β,γ-methylene)triphosphate (GPPCP), p3-1-(2-nitro)phenylethylguanosine- 5'-triphosphate (caged GTP) and Elongation Factor Ts. An attempt to crystallize a ternary complex of EF-Tu·GPPNP with Phe-tRNAPhe was made. The crystals of EF-Tu obtained with GPPNP or GPPCP, respectively, were suitable for X-ray diffraction analysis at very high resolution.

  • Crystals of intact Elongation Factor Tu from Thermus thermophilus diffracting to high resolution.
    Journal of molecular biology, 1991
    Co-Authors: L.s. Reshetnikova, Christian O. A. Reiser, Norbert K. Schirmer, H. Berchtold, R. Storm, Rolf Hilgenfeld, Mathias Sprinzl
    Abstract:

    Abstract The intact Elongation Factor Tu from the extreme thermophile Thermus thermophilus has been crystalized as a complex with the GTP analogue guanosine-5′-(β-γ-imido)triphosphate. The crystals are very stable in the X-ray beam and diffract to 1·9 A resolution. They exhibit space group C2, with a = 150·(36) A , b = 99·6(3) A , c = 40·1(1) A , β = 95·4(2)°, and contain one Elongation Factor Tu molecule per asymmetric unit.

  • Effect of Thermus thermophilus Elongation Factor Ts on the conformation of Elongation Factor Tu
    European journal of biochemistry, 1991
    Co-Authors: Norbert K. Schirmer, Christian O. A. Reiser, Mathias Sprinzl
    Abstract:

    Affinity labeling in situ of the Thermus thermophilus Elongation Factor Tu (EF-Tu) nucleotide binding site was achieved with periodate-oxidized GDP (GDPoxi) or GTP (GTPoxi) in the absence and presence of Elongation Factor Ts (EF-Ts). Lys52 and Lys137, both reacting with GDPoxi and GTPoxi, are located in the nucleotide binding region. In the absence of EF-Ts Lys137 and to a lesser extent Lys52 were accessible to the reaction with GTPoxi, GDPoxi reacted much more efficiently with Lys52 than with Lys137 under these conditions [Peter, M. E., Wittman-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132–9138]. In the presence of EF-Ts, GDPoxi reacted more efficiently with Lys137 than with Lys52, indicating that the interaction of EF-Ts with EF-Tu˙ GDPoxi induces a conformation resembling that of the EF-Tu˙ GTPoxi complex in the absence of EF-Ts. Binding of EF-Ts to EF-Tu˙ GDP enhances the accessibility of the Arg59-Gly60 peptide bond of EF-Tu to trypsin cleavage. Hydrolysis of this peptide bond does not interfere with the ability of EF-Ts to bind to EF-Ts. EF-Ts is protected against trypsin cleavage by interaction with EF-Tu˙ GDP. High concentrations of EF-Ts did not interfere significantly with aminoacyl-tRNA˙ EF–Tu˙ GTP complex formation.