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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.

  • 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.

  • Elongation Factor Tu: a regulatory GTPase with an integrated effector
    Trends in biochemical sciences, 1994
    Co-Authors: Mathias Sprinzl
    Abstract:

    Abstract Several Elongation Factors involved in protein synthesis are GTPases that share strucTural and mechanistic homology with the large family of proteins including Ras and heterotrimeric receptor-coupled G proteins. The strucTure of Elongation Factor Tu (EF-Tu) from thermophilic bacteria, in its ‘active' GTP-bound form, has recently been solved by X-ray crystallography. Comparison of this strucTure with the strucTure of Escherichia coli EF-Tu bound to GDP reveals a dramatic conformational change that is dependent on GTPase activity. The mechanism of this conformational change and of GTPase activation are discussed, and a model for the EF-Tu-GTP complex with aminoacyl-tRNA is presented.

  • Crystal strucTure of active Elongation Factor Tu reveals major domain rearrangements
    Nature, 1993
    Co-Authors: Harald Berchtold, Mathias Sprinzl, Christian O. A. Reiser, Norbert K. Schirmer, Ludmila Reshetnikova, Rolf Hilgenfeld
    Abstract:

    The crystal strucTure of intact Elongation Factor Tu (EF-Tu) from Thermus thermophilus has been determined and refined at an effective resolution of 1.7 A, with incorporation of data extending to 1.45 A. The effector region, including interaction sites for the ribosome and for transfer RNA, is well defined. Molecular mechanisms are proposed for transductlon and amplification of the signal induced by GTP binding as well as for the intrinsic and effector-enhanced GTPase activity of EF-Tu. Comparison of the strucTure with that of EF-Tu–GDP reveals major muTual rearrange-ments of the three domains of the molecule.

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

  • The busiest of all ribosomal assistants: Elongation Factor Tu.
    Biochemistry, 2012
    Co-Authors: Darius Kavaliauskas, Poul Nissen, Charlotte R. Knudsen
    Abstract:

    During translation, the nucleic acid language employed by genes is translated into the amino acid language used by proteins. The translator is the ribosome, while the dictionary employed is known as the genetic code. The genetic information is presented to the ribosome in the form of a mRNA, and tRNAs connect the two languages. Translation takes place in three steps: initiation, Elongation, and termination. After a protein has been synthesized, the components of the translation apparaTus are recycled. During each phase of translation, the ribosome collaborates with specific translation Factors, which secure a proper balance between speed and fidelity. Notably, initiation, termination, and ribosomal recycling occur only once per protein produced during normal translation, while the Elongation step is repeated a large number of times, corresponding to the number of amino acids constiTuting the protein of interest. In bacteria, Elongation Factor Tu plays a central role during the selection of the correct ami...

  • Isolation of Qbeta polymerase complexes containing mutant species of Elongation Factor Tu.
    Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2003
    Co-Authors: Sander G J Mathu, Charlotte R. Knudsen, Jan Van Duin, Barend Kraal
    Abstract:

    The RNA genome of coliphage Qbeta is replicated by a complex of four proteins, one of them being the translation Elongation Factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.

  • Isolation of Qβ polymerase complexes containing mutant species of Elongation Factor Tu
    Journal of Chromatography B, 2002
    Co-Authors: Sander G J Mathu, Charlotte R. Knudsen, Jan Van Duin, Barend Kraal
    Abstract:

    Abstract The RNA genome of coliphage Qβ is replicated by a complex of four proteins, one of them being the translation Elongation Factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.

  • 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.

Brian F.c. Clark - 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.

  • Site-directed mutagenesis of Arg58 and Asp86 of Elongation Factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding
    Protein engineering, 1995
    Co-Authors: Charlotte R. Knudsen, Brian F.c. Clark
    Abstract:

    Elongation Factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of Elongation Factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination of the dissociation and association rate constants. The results imply that Arg58 is unimportant for the intrinsic GTPase mechanism and the binding of guanine nucleotides, whereas it is strongly involved in the binding of aminoacyl-tRNA and of the ribosome. Asp86 appears to be essential for the regulation of guanine-nucleotide affinities, and it may also play a role in the intrinsic GTPase mechanism.

  • Towards an understanding of strucTure-function relationships of Elongation Factor Tu
    Biotechnology and applied biochemistry, 1994
    Co-Authors: Ove Wiborg, Charlotte R. Knudsen, Carsten Andersen, T. J. Kristensen, Brian F.c. Clark
    Abstract:

    In light of the recently determined strucTure of Elongation Factor Tu, and taking into account chemical sTudies mapping functional sites, a number of residues have been selected for site-directed mutagenesis sTudies. Gly 94 , Gly 126 , His 66 , His 118 , Lys 89 and Asp 90 have each been point-mutated. Preliminary in vitro characterization data are presented

Andrea Parmeggiani - One of the best experts on this subject based on the ideXlab platform.

  • Elongation Factor Tu targeted antibiotics four different strucTures two mechanisms of action
    FEBS Letters, 2006
    Co-Authors: Andrea Parmeggiani, Poul Nissen
    Abstract:

    Elongation Factor Tu (EF-Tu), the carrier of aa-tRNA to the mRNA-programmed ribosome, is the target of four families of antibiotics of unrelated strucTure, of which the action is supported by two basic mechanisms. Kirromycin and enacyloxin block EF-Tu · GDP on the ribosome; pulvomycin and GE2270 A inhibit the interaction of EF-Tu · GTP with aa-tRNA. The crystallographic analysis has unveiled the strucTural background of their actions, explaining how antibiotics of unrelated strucTures and binding modes and sites can employ similar mechanism of action. The selective similarities and differences of their binding sites and the induced EF-Tu conformations make understand how naTure can affect the activities of a complex regulatory enzyme by means of low-molecular compounds, and have proposed a suitable approach for drug design.

  • SITE-DIRECTED MUTAGENESIS OF Elongation Factor Tu
    European journal of biochemistry, 1992
    Co-Authors: Pieter H. Anborgh, Andrea Parmeggiani, Jiří Jonák
    Abstract:

    Elongation Factor Tu (EF-Tu), a monomeric protein of 393 amino acid residues (M.W. 43,000), is the most abundant protein in E.coli and one of the best sTudied guanine nucleotide binding proteins, a family of enzymes involved in signal transduction in higher and lower organisms (for references see Bosch et al., 1984; Gilman, 1984; Parmeggiani and Swart, 1985; Bourne, 1986). These proteins bind GTP and GDP, are able to hydrolyze GTP and show typical homologies in their primary strucTures, especially in the N-terminal 150–200 amino acids. The finding that the ras p21 protein, a mutant variant of which is responsible for oncogenic transformation, is a guanine nucleotide binding protein (Scolnick et al., 1979) further emphasizes the importance of this family. EF-Tu is an essential component of protein biosynthesis, acting as the carrier of aa-tRNA to the ribosome (for references, see Milled: & Weissbach, 1977). As with the other guanine nucleotide binding proteins, GTP induces the active form of the Factor: only EF-Tu·GTP is capable of interacting with aa-tRNA, forming a ternary complex.

  • Elongation Factor Tu: a molecular switch in protein biosynthesis
    Molecular microbiology, 1992
    Co-Authors: Albert Weijland, Robbert H. Cool, Pieter H. Anborgh, Kim Harmark, Andrea Parmeggiani
    Abstract:

    Elongation Factor Tu (EF-Tu), the most abundant protein in Escherichia coli, is a guanine nucleotide-binding protein that in the 'on' state acts as a carrier of amino acyl-tRNA to the ribosome. Our knowledge of this essential component of translation has brought substantial progress in the past decade thanks to the co-ordinated application of biochemical, physico-chemical and genetic methods. Crystallographic analysis at 2.6 A resolution and site-directed mutagenesis have revealed strucTural and functional similarities between the guanine nucleotide-binding domains of EF-Tu and human H-ras p21 protein. The regulation of the expression of the two EF-Tu-encoding genes in E. coli, particularly that of TufB, has been shown to involve diverse mechanisms. Several aspects of the functions of EF-Tu in the Elongation cycle have been reinvestigated, leading to new insights. These sTudies have emphasized the manifold aspects of the mechanisms regulating the activity of EF-Tu in the bacterial cell.

  • SubstiTution of proline 82 by threonine induces autophosphorylating activity in GTP-binding domain of Elongation Factor Tu.
    The Journal of biological chemistry, 1990
    Co-Authors: Robbert H. Cool, Brian F.c. Clark, Michael D. Jensen, J. Jonak, Andrea Parmeggiani
    Abstract:

    Abstract Mutation of Pro82 into Thr, a residue siTuated in the second element (D80CPG83) of the consensus sequence proposed to interact with GTP/GDP in GTP-binding proteins was introduced via site-directed mutagenesis in the isolated guanine nucleotide-binding domain (G domain) of Elongation Factor Tu. G domainPT82 displays virTually no GTPase activity. As a major change, the apparent inhibition of the GTPase reaction is associated with the appearance of autophosphorylating activity, as in ras product p21 in the case of mutation Ala59----Thr, corresponding to 82 in Elongation Factor Tu. Dependence of this reaction on mono- and divalent cation concentration and on pH is essentially the same as for the GTPase of wild-type G domain. The autokinase reaction follows an apparent first order rate, suggesting an intermolecular mechanism. Analysis of amino acid and peptide composition of the 32P-labeled G domainPT82, as well as Edman degradation of the tryptic peptide containing the covalently bound 32P, shows that Thr82 is the phosphorylated residue. Taken together, these results point out that Thr82 is in close proximity to the gamma-phosphate of GTP, as in the case of Thr59 in p21. These results are in agreement with the observations derived from x-ray diffraction analysis that the tertiary strucTure of the GTP-binding domain of Elongation Factor Tu and that of p21 are similar.

Rolf Hilgenfeld - One of the best experts on this subject based on the ideXlab platform.

  • Inhibitory Mechanisms of Antibiotics Targeting Elongation Factor Tu
    Current protein & peptide science, 2002
    Co-Authors: Tanis Hogg, Jeroen R. Mesters, Rolf Hilgenfeld
    Abstract:

    Since the pioneering discovery of the inhibitory effects of kirromycin on bacterial Elongation Factor Tu (EF-Tu) more than 25 years ago [1], a great wealth of biological data has accumulated concerning protein biosynthesis inhibitors specific for EF-Tu. With the subsequent discovery of over two dozen naTurally occurring EF-Tu inhibitors belonging to four different subclasses, EF-Tu has blossomed into an appealing antimicrobial target for rational drug discovery efforts. Very recently, independent crystal strucTure determinations of EF-Tu in complex with two potent antibiotics, aurodox and GE2270A, have provided strucTural explanations for the mode of action of these two compounds, and have set the foundation for the design of inhibitors with higher bioavailability, broader spectra, and greater efficacy.

  • phosphorylation of Elongation Factor Tu prevents ternary complex formation
    Journal of Biological Chemistry, 1995
    Co-Authors: Christian Alexander, Barend Kraal, Rolf Hilgenfeld, Jeroen R. Mesters, Nese Bilgin, Carsten Lindschau, Volker A Erdmann, Corinna Lippmann
    Abstract:

    Abstract The Elongation Factor Tu (EF-Tu) is a member of the GTP/GDP-binding proteins and interacts with various partners during the Elongation cycle of protein biosynthesis thereby mediating the correct binding of aminoacylated transfer RNA (aa-tRNA) to the acceptor site (A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its GDP-bound state. In vivo, EF-Tu is post-translationally modified by phosphorylation. Here we report that the phosphorylation of EF-Tu by a ribosome associated kinase activity is drastically enhanced by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function, inhibits the modification. This effect is specific, since kirromycin-resistant mutants do become phosphorylated in the presence of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu does not bind kirromycin. Most interestingly, the phosphorylation of EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation the site of modification is located at the interface between domains 1 and 3 and is involved in a strong interdomain hydrogen bond. Introduction of a charged phosphate group at this position will change the interaction between the domains, leading to an opening of the molecule reminiscent of the GDP conformation. A model for the function of EF-Tu phosphorylation in protein biosynthesis is presented.

  • Crystal strucTure of active Elongation Factor Tu reveals major domain rearrangements
    Nature, 1993
    Co-Authors: Harald Berchtold, Mathias Sprinzl, Christian O. A. Reiser, Norbert K. Schirmer, Ludmila Reshetnikova, Rolf Hilgenfeld
    Abstract:

    The crystal strucTure of intact Elongation Factor Tu (EF-Tu) from Thermus thermophilus has been determined and refined at an effective resolution of 1.7 A, with incorporation of data extending to 1.45 A. The effector region, including interaction sites for the ribosome and for transfer RNA, is well defined. Molecular mechanisms are proposed for transductlon and amplification of the signal induced by GTP binding as well as for the intrinsic and effector-enhanced GTPase activity of EF-Tu. Comparison of the strucTure with that of EF-Tu–GDP reveals major muTual rearrange-ments of the three domains of the molecule.

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