PubMed Health⌕ Search

Biomedical subjects

R Giegé

Publications and source records attributed to R Giegé.

At least 55 records · Page 3Linked to original sources
PubMed1998

Sequences outside recognition sets are not neutral for tRNA aminoacylation. Evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe.

Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363-1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia coli tRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a "classical" recognition element since its antideterminant effect is compensated for by a second-site mutation. This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name "permissive elements," are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently.

Base Sequence↗
PubMed1998

The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA.

Direct sequencing of human mitochondrial tRNALysshows the absence of editing and the occurrence of six modified nucleotides (m1A9, m2G10, Psi27, Psi28 and hypermodified nucleotides at positions U34 and A37). This tRNA folds into the expected cloverleaf, as confirmed by structural probing with nucleases. The solution structure of the corresponding in vitro transcript unexpectedly does not fold into a cloverleaf but into an extended bulged hairpin. This non-canonical fold, established according to the reactivity to a large set of chemical and enzymatic probes, includes a 10 bp aminoacyl acceptor stem (the canonical 7 bp and 3 new pairs between residues 8-10 and 65-63), a 13 nt large loop and an anticodon-like domain. It is concluded that modified nucleotides have a predominant role in canonical folding of human mitochondrial tRNALys. Phylogenetic comparisons as well as structural probing of selected in vitro transcribed variants argue in favor of a major contribution of m1A9 in this process.

Cloning, Organism↗
PubMed1998

Transfer RNA identity rules and conformation of the tyrosine tRNA-like domain of BMV RNA imply additional charging by histidine and valine.

This paper reports the first example of a triple aminoacylation specificity of a viral tRNA-like domain. These findings were based on structural studies on the brome mosaic virus (BMV) tRNA-like domain (Felden et al., 1994, J. Mol. Biol. 235, 508-531) together with knowledge on tRNA aminoacylation identity rules suggesting potential histidinylation and valylation capacities of the viral RNA in addition to its already known tyrosylation ability. Here, both predictions are demonstrated by in vitro aminoacylation assays. Kinetic parameters of histidinylation and valylation of BMV tRNA-like structure have been determined and compared to those of the corresponding tRNA transcripts and to the tyrosylation capacity of the molecule. The influence of experimental conditions on aminoacylation reactions was also studied. The novel aminoacylation capacities of BMV tRNA-like domain support its already reported three-dimensional fold and illustrate the predictive potential of modeling data. Biological necessity of specific or non specific aminoacylation will be discussed.

Base Sequence↗
PubMed1998

Glycyl-tRNA synthetase from Thermus thermophilus--wide structural divergence with other prokaryotic glycyl-tRNA synthetases and functional inter-relation with prokaryotic and eukaryotic glycylation systems.

The tRNA glycylation system is amongst the most complex aminoacylation systems since neither the oligomeric structure of the enzymes nor the discriminator base in tRNAs are conserved in the phylae. To understand better this structural diversity and its functional consequences, the prokaryotic glycylation system from Thermus thermophilus, an extreme thermophile, was investigated and its structural and functional inter-relations with those of other origins analyzed. Alignments of the protein sequence of the dimeric thermophilic glycyl-tRNA synthetase (Gly-tRNA synthetase) derived from its gene with sequences of other dimeric Gly-tRNA synthetases revealed an atypical character of motif 1 in all these class 2 synthetases. Interestingly, the sequence of the prokaryotic thermophilic enzyme resembles eukaryotic and archaebacterial Gly-tRNA synthetases, which are all dimeric, and diverges drastically from the tetrameric enzymes from other prokaryotes. Cross aminoacylations with tRNAs and synthetases of different origins provided information about functional interrelations between the glycylation systems. Efficient glycylations involving partners from T. thermophilus and Escherichia coli showed conservation of the recognition process in prokaryotes despite strong structural variations of the synthetases. However, Gly-tRNA synthetase from T. thermophilus acylates eukaryotic tRNA(Gly) while the charging ability of the E. coli enzyme is restricted to prokaryotic tRNA(Gly). A similar behaviour is found in eukaryotic systems where the restricted species specificity for tRNA glycylation of mammalian Gly-tRNA synthetase contrasts with the relaxed specificity of the yeast enzyme. The consensus sequence of the tRNAs charged by the various Gly-tRNA synthetases reveals conservation of only G1-C72 in the acceptor arm, C35 and C36 in the anticodon, and the (G10-Y25)-G45 triplet involved in tRNA folding. Conservation of these nucleotides indicates their key role in glycylation and suggests that they were part of the ancestral glycine identity set. These features are discussed in the context of the phylogenic connections between prokaryotes, eukaryotes, and archaebacteria, and of the particular place of T. thermophilus in this phylogeny.

Amino Acid Sequence↗
PubMed1998

Effect of a mutation in the anticodon of human mitochondrial tRNAPro on its post-transcriptional modification pattern.

Although the gene sequences of all 22 tRNAs encoded in the human mitochondrial genome are known, little information exists about their sequences at the RNA level. This becomes a crucial limitation when searching for a molecular understanding of the growing number of maternally inherited human diseases correlated with point mutations in tRNA genes. Here we describe the sequence of human mt-tRNAPropurified from placenta. It shows absence of editing events in this tRNA and highlights the presence of eight post-transcriptional modifications. These include T54, never found so far in an animal mt-tRNA, and m1G37, a modification known to have fundamental functional properties in a number of canonical tRNAs. Occurrence of m1G37 was further investigated in an analysis of the substrate properties of in vitro transcripts of human mt-tRNAProtowards pure Escherichia coli methylguanosine transferase. This enzyme properly methylates G37 in mt-tRNA and is sensitive to the presence of a second G at position 36, neighboring the target nucleotide for methylation. Since mutation of nt 36 was shown to be correlated with myopathy, the potential consequences of non-modification or under-modification of mt-tRNA nucleotides in expression of the particular myopathy and of mitochondrial diseases in general are discussed.

Anticodon↗
PubMed1998

A pseudoknotted tRNA variant is a substrate for tRNA (cytosine-5)-methyltransferase from Xenopus laevis.

tRNA post-transcriptional modification enzymes of Xenopus laevis were proposed previously to belong to two major groups according to their sensitivity to structural perturbations in their substrates. To further investigate the structural variations tolerated by these enzymes, the tRNA-like domain of turnip yellow mosaic virus RNA (88 nucleotides in length) has been microinjected into the oocytes of Xenopus laevis. This RNA possesses 12 potential target nucleotides for modification within a structure including a pseudoknotted folding, an extended anticodon stem, and unusual D-loop/T-loop interactions. Results indicate that only cytosine-42, a position equivalent to C-49 in canonical tRNAs, was quantitatively modified into m5C in the microinjected RNA. Modification was detected to high levels, indicating that at least one enzyme tolerates non-canonical structural features.

Animals↗
PubMed1998

tRNA mimics.

Mimics recapitulating the structural features of tRNAs are involved in biological processes other than ribosome-dependent protein synthesis. A knowledge of the rules underlying the architecture and function of tRNAs allows the design of non-natural mimics. The study of these mimics sheds light upon links between replication, translation and metabolic pathways, leads to biotechnological applications, and provides experimental and conceptual tools for the exploration of primordial evolutionary processes.

Animals↗
PubMed1998

The RNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA.

Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNA(Arg) and C36 and G37 in tRNA(Asp) (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069-5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNA(Arg) and tRNA(Asp), the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899-4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different k(cat) and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system.

Anticodon↗
PubMed1998

Isoleucylation properties of native human mitochondrial tRNAIle and tRNAIle transcripts. Implications for cardiomyopathy-related point mutations (4269, 4317) in the tRNAIle gene.

A growing number of mutated mitochondrial tRNA genes have been found associated with severe human diseases. To investigate the potential interference of such mutations with the primordial function of tRNAs, i.e. their aminoacylation by cognate aminoacyl-tRNA synthetases, a human mitochondrial in vitro aminoacylation system specific for isoleucine has been established. Both native tRNAIleand isoleucyl-tRNA synthetase activity have been recovered from human placental mitochondria and the kinetic parameters of tRNA aminoacylation determined. The effect of pathological point mutations present in the mitochondrial gene encoding tRNAIlehas been tackled by investigating the isoleucylation properties of wild-type and mutated in vitro transcripts. Data show that: (i) modified nucleotides contribute to efficient isoleucylation; (ii) point mutation A4269G in the gene (A-->G at nt 7 in the tRNA), associated with a cardiomyopathy, does not affect aminoacylation significantly; (iii) point mutation A4317G (A-->G at nt 59 in the tRNA), reported in a case of fatal infantile cardiomyopathy, induces a small but significant decrease in isoleucylation. The potential implications of these findings on the understanding of the molecular mechanisms involved in the expression of pathology are discussed.

Adenine↗
PubMed1997

Mirror image alternative interaction patterns of the same tRNA with either class I arginyl-tRNA synthetase or class II aspartyl-tRNA synthetase.

Gene cloning, overproduction and an efficient purification protocol of yeast arginyl-tRNA synthetase (ArgRS) as well as the interaction patterns of this protein with cognate tRNAArgand non-cognate tRNAAspare described. This work was motivated by the fact that the in vitro transcript of tRNAAspis of dual aminoacylation specificity and is not only aspartylated but also efficiently arginylated. The crystal structure of the complex between class II aspartyl-tRNA synthetase (AspRS) and tRNAAsp, as well as early biochemical data, have shown that tRNAAspis recognized by its variable region side. Here we show by footprinting with enzymatic and chemical probes that transcribed tRNAAspis contacted by class I ArgRS along the opposite D arm side, as is homologous tRNAArg, but with idiosyncratic interaction patterns. Besides protection, footprints also show enhanced accessibility of the tRNAs to the structural probes, indicative of conformational changes in the complexed tRNAs. These different patterns are interpreted in relation to the alternative arginine identity sets found in the anticodon loops of tRNAArgand tRNAAsp. The mirror image alternative interaction patterns of unmodified tRNAAspwith either class I ArgRS or class II AspRS, accounting for the dual identity of this tRNA, are discussed in relation to the class defining features of the synthetases. This study indicates that complex formation between unmodified tRNAAspand either ArgRS and AspRS is solely governed by the proteins.

Anticodon↗
PubMed1997

Comparative analysis of thaumatin crystals grown on earth and in microgravity.

The protein thaumatin was studied as a model macromolecule for crystallization in microgravity-environment experiments conducted on two US Space Shuttle missions (USML-2 and LMS). In this investigation, we have evaluated and compared the quality of space- and earth-grown thaumatin crystals using X-ray diffraction analyses, and characterized them according to crystal size, diffraction resolution limit and mosaicity. Two different approaches for growing thaumatin crystals in the microgravity environment, dialysis and liquid-liquid diffusion, were employed as a joint experiment by our two investigative teams. Thaumatin crystals grown in a microgravity environment were generally larger in volume and the total number of crystals was less, relative to crystals grown on earth. They diffracted to significantly higher resolution and with improved diffraction properties, as judged by relative plots of I/sigma versus resolution. The mosaicity of space-grown crystals was significantly less than that of crystals grown on earth. Increased concentrations of protein in the crystallization chambers in microgravity led to larger crystals. The data presented here lend further support to the idea that protein crystals of improved quality can be obtained in a microgravity environment.

Journal Article↗
PubMed1997

Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus. Structural and biochemical properties of the two enzymes.

Two aspartyl-tRNA synthetases (AspRSs) were isolated from Thermus thermophilus HB8. Both are alpha2 dimers but differ in the length of their polypeptide chains (AspRS1, 68 kDa; and AspRS2, 51 kDa). Both chains start with Met and are deprived of common sequences to a significant extent. This rules out the possibility that AspRS2 is derived from AspRS1 by proteolysis, in agreement with specific recognition of each AspRS by the homologous antibodies. DNA probes derived from N-terminal amino acid sequences hybridize specifically to different genomic DNA fragments, revealing that the two AspRSs are encoded by distinct genes. Both enzymes are present in various strains from T. thermophilus and along the growth cycle of the bacteria, suggesting that they are constitutive. Kinetic investigations show that the two enzymes are specific for aspartic acid activation and tRNAAsp charging. tRNA aspartylation by the thermostable AspRSs is governed by thermodynamic parameters which values are similar to those measured for mesophilic aspartylation systems. Both thermophilic AspRSs are deprived of species specificity for tRNA aspartylation and exhibit N-terminal sequence signatures found in other AspRSs, suggesting that they are evolutionarily related to AspRSs from mesophilic prokaryotes and eukaryotes. Comparison of the efficiency of tRNA aspartylation by each enzyme under conditions approaching the physiological ones suggests that in vivo tRNAAsp charging is essentially ensured by AspRS1, although AspRS2 is the major species. The physiological significance of the two different AspRSs in T. thermophilus is discussed.

Amino Acid Sequence↗
PubMed1997

Visualization of RNA crystal growth by atomic force microscopy.

The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.

Crystallization↗
PubMed1997

Rapid selection of aminoacyl-tRNAs based on biotinylation of alpha-NH2 group of charged amino acids.

A rapid selection procedure to separate low amounts of aminoacylated tRNAs from large pools of inactive variants is described. The procedure involves a three-step protocol. After initial aminoacylation of a tRNA pool, N-hydroxysuccinimide ester chemistry is applied to biotinylate the alpha-NH2 group of the amino acid bound to the 3'-end of a tRNA. The biotin tag is used to capture the derivatized tRNAs on streptavidin-conjugated magnetic beads. Variants bound to the solid phase can be amplified by RT-PCR and transcription, providing tRNAs for subsequent selection rounds.

Amino Acids↗
PubMed1997

Strategy for RNA recognition by yeast histidyl-tRNA synthetase.

Histidine aminoacylation systems are of interest because of the structural diversity of the RNA substrates recognized by histidyl-tRNA synthetases. Among tRNAs participating in protein synthesis, those specific for histidine all share an additional residue at their 5'-extremities. On the other hand, tRNA-like domains at the 3'--termini of some plant viruses can also be charged by histidyl-tRNA synthetases, although they are not actors in protein synthesis. This is the case for the RNAs from tobacco mosaic virus and its satellite virus but also those of turnip yellow and brome mosaic viruses. All these RNAs have intricate foldings at their 3'-termini differing from that of canonical tRNAs and share a pseudoknotted domain which is the prerequisite for their folding into structures mimicking the overall L-shape of tRNAs. This paper gives an overview on tRNA identity and rationalizes the apparently contradictory structural and aminoacylation features of histidine-specific tRNAs and tRNA-like structures. The discussion mainly relies on histidylation data obtained with the yeast synthetase, but the conclusions are of a more universal nature. In canonical tRNA(His), the major histidine identity element is the 'minus' 1 residue, since its removal impairs histidylation and conversely its addition to a non-cognate tRNA(Asp) confers histidine identity to the transplanted molecule. Optimal expression of histidine identity depends on the chemical nature of the -1 residue and is further increased and/or modulated by the discriminator base N73 and by residues in the anticodon. In the viral tRNA-like domains, the major identity determinant -1 is mimicked by a residue from the single-stranded L1 regions of the different pseudoknots. The consequences of this mimicry for the function of minimalist RNAs derived from tRNA-like domains are discussed. The characteristics of the histidine systems illustrate well the view that the core of the amino acid accepting RNAs is a scaffold that allows proper presentation of identity nucleotides to their amino acid identity counterparts in the synthetase and that different types of scaffoldings are possible.

Base Sequence↗
PubMed1997

Structure and aminoacylation capacities of tRNA transcripts containing deoxyribonucleotides.

The contribution of the ribose 2'-hydroxyls to RNA structure and function has been analyzed, but still remains controversial. In this work, we report the use of a mutant T7 RNA polymerase as a tool in RNA studies, applied to the aspartate and methionine tRNA aminoacylation systems from yeast. Our approach consists of determining the effect of substituting natural ribonucleotides by deoxyribonucleotides in RNA and, thereby, defining the subset of important 2'-hydroxyl groups. We show that deoxyribose-containing RNA can be folded in a global conformation similar to that of natural RNA. Melting curves of tRNAs, obtained by temperature-gradient gel electrophoresis, indicate that in deoxyribo-containing molecules, the thermal stability of the tertiary network drops down, whereas the stability of the secondary structure remains unaltered. Nuclease footprinting reveals a significant increase in the accessibility of both single- and double-stranded regions. As to the functionality of the deoxyribose-containing tRNAs, their in vitro aminoacylation efficiency indicates striking differential effects depending upon the nature of the substituted ribonucleotides. Strongest decrease in charging occurs for yeast initiator tRNA(Met) transcripts containing dG or dC residues and for yeast tRNA(Asp) transcripts with dU or dG. In the aspartate system, the decreased aminoacylation capacities can be correlated with the substitution of the ribose moieties of U11 and G27, disrupting two hydrogen bond contacts with the synthetase. Altogether, this suggests that specific 2'-hydroxyl groups in tRNAs can act as determinants specifying aminoacylation identity.

Base Sequence↗
PubMed1996

An example of non-conservation of oligomeric structure in prokaryotic aminoacyl-tRNA synthetases. Biochemical and structural properties of glycyl-tRNA synthetase from Thermus thermophilus.

Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D.T., Mazauric, M.-H., Kern, D. & Moras, D. (1995) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha 2 type consistent with that found for eukaryotic Gly-tRNA synthetases (yeast and Bombyx mori), but different from that of Gly-tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are alpha 2 beta 2 tetramers. N-terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenzae and Coxiella burnetii) albeit the enzyme is deprived of the N-terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly-tRNA synthetase combines strong structural homologies of eukaryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-PPi exchange and aminoacylation activities up to 70 degrees C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA(Gly) does not induce protection. Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA(gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(GCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity. The results are discussed in the context of the three-dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.

Amino Acid Sequence↗