Biomedical subjects

R Giegé

Publications and source records attributed to R Giegé.

At least 73 records · Page 4Linked to original sources

PubMed1996

Arginine aminoacylation identity is context-dependent and ensured by alternate recognition sets in the anticodon loop of accepting tRNA transcripts.

Yeast arginyl-tRNA synthetase recognizes the non-modified wild-type transcripts derived from both yeast tRNA(Arg) and tRNA(Asp) with equal efficiency. It discriminates its cognate natural substrate, tRNA(Arg), from non-cognate tRNA(Asp) by a negative discrimination mechanism whereby a single methyl group acts as an anti-determinant. Considering these facts, recognition elements responsible for specific arginylation in yeast have been searched by studying the in vitro arginylation properties of a series of transcripts derived from yeast tRNA(Asp), considered as an arginine isoacceptor tRNA. In parallel, experiments on similar tRNA(Arg) transcripts were performed. Unexpectedly, in the tRNA(Arg) context, arginylation is basically linked to the presence of residue C35, whereas in the tRNA(Asp) context, it is deeply related to that of C36 and G37 but is insensitive to the nucleotide at position 35. Each of these nucleotides present in one host, is absent in the other host tRNA. Thus, arginine identity is dependent on two different specific recognition sets according to the tRNA framework investigated.

Anticodon↗

PubMed1996

Fe.bleomycin as a probe of RNA conformation.

Two crystallographically defined tRNAs, yeast tRNAAsp and tRNAPhe, were used as substrates for oxidative cleavage by Fe.bleomycin to facilitate definition at high resolution of the structural elements in RNAs conducive to bleomycin binding and cleavage. Yeast tRNAAsp underwent cleavage at G45 and U66; yeast tRNAPhe was cleaved at four sites, namely G19, A31, U52 and A66. Only two of these six sites involved oxidative cleavage of a 5'-G.Pyr-3' sequence, but three sites were at the junction between single- and double-stranded regions of the RNA, consistent with a binding model in which the bithiazole + C-terminal substituent of bleomycin bind to minor groove structures on the RNA. Also studied were four tRNA transcripts believed on the basis of biochemical and chemical mapping experiments to share structural elements in common with the mature tRNAs. Cleavage of these tRNAs by Fe.bleomycin gave patterns of cleavage very different from each other and than those of the mature tRNAs. This observation suggests strongly that Fe.bleomycin cannot be used for chemical mapping in the same fashion as more classical reagents, such as Pb2+ or dimethyl sulfate. However, the great sensitivity of Fe.bleomycin to changes in nucleic acid structure argues that those species which do show similar patterns of cleavage must be very close in structure.

Binding Sites↗

PubMed1996

Identity of prokaryotic and eukaryotic tRNA(Asp) for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus.

The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements. It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS. This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems. Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs. It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions. Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T. thermophilus AspRS. In the homologous systems, the mutational effects are less pronounced. However, effects in E. coli and T. thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes. Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex.

Amino Acid Sequence↗

PubMed1996

Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.

Base Sequence↗

PubMed1996

Enzymatic formation of modified nucleosides in tRNA: dependence on tRNA architecture.

Information is still quite limited concerning the structural requirements in tRNA molecules for their post-transcriptional maturation by base and ribose modification enzymes. To address this question, we have chosen as the model system yeast tRNAAsp that has a known three-dimensional structure and the in vivo modifying machinery of the Xenopus laevis oocyte able to act on microinjected tRNA precursors. We have systematically compared the modification pattern of wild-type tRNAAsp with that of a series of structural mutants (21 altogether) altered at single or multiple positions in the D-, T-and the anticodon branch, as well as in the variable region. The experimental system allowed us to analyze the effects of structural perturbations in tRNA on the enzymatic formation of modified nucleosides at 12 locations scattered over the tRNA cloverleaf. We found that the formation of m1G37 and psi 40 in the anticodon loop and stem and psi 13 in the D-stem, were extremely sensitive to 3D perturbations. In contrast, the formation of T54, psi 55 and m1A58 in the T-loop, m5C49 in the T-stem and m2G6 in the amino acid accepting stem were essentially insensitive to change in the overall tRNA architecture; these modified nucleosides were also formed in appropriate minimalist (stems and loops) tRNA domains. The formation of m2G26 at the junction between the anticodon and the D-stem, of Q34 and manQ34 in the anticodon loop were sensitive only to drastic structural perturbation of the tRNA. Altogether, these results reflect the existence of different modes of tRNA recognition by the many different modifying enzymes. A classification of this family of maturation enzymes into two major groups, according to their sensitivities to structural perturbations in tRNA, is proposed.

Animals↗

PubMed1996

Usefulness of functional and structural solution data for the modeling of tRNA-like structures.

Structures of large RNAs are not easily solved by X-ray crystallography or by NMR spectroscopy. This paper reviews the alternate methodology based on enzymatic and chemical mapping data collected on RNAs combined with graphical modeling for the construction of three-dimensional models. The different steps that lead to the establishment of the models are critically discussed. It is shown how the correctness of an RNA model can be strengthened by establishing correlations between the structure and the functionality of the molecule and its variants. Finally, the predictive potential of a model is discussed The approach is illustrated by results obtained on plant viral tRNA-like structures, and particularly on that of brome mosaic virus (BMV) RNA.

Base Sequence↗

PubMed1996

Aspartate identity of transfer RNAs.

Structure/function relationships accounting for specific tRNA charging by class II aspartyl-tRNA synthetases from Saccharomyces cerevisiae, Escherichia coli and Thermus thermophilus are reviewed. Effects directly linked to tRNA features are emphasized and aspects about synthetase contribution in expression of tRNA(Asp) identity are also covered. Major identity nucleotides conferring aspartate specificity to yeast, E coli and T thermophilus tRNAs comprise G34, U35, C36, C38 and G73, a set of nucleotides conserved in tRNA(Asp) molecules of other biological origin. Aspartate specificity can be enhanced by negative discrimination preventing, eg mischarging of native yeast tRNA(Asp by yeast arginyl-tRNA synthetase. In the yeast system crystallography shows that identity nucleotides are in contact with identity amino acids located in the catalytic and anticodon binding domains of the synthetase. Specificity of RNA/protein interaction involves a conformational change of the tRNA that optimizes the H-bonding potential of the identity signals on both partners of the complex. Mutation of identity nucleotides leads to decreased aspartylation efficiencies accompanied by a loss of specific H-bonds and an altered adaptation of tRNA on the synthetase. Species-specific characteristics of aspartate systems are the number, location and nature of minor identity signals. These features and the structural variations in aspartate tRNAs and synthetases are correlated with mechanistic differences in the aminoacylation reactions catalyzed by the various aspartyl-tRNA synthetases. The reality of the aspartate identity set is verified by its functional expression in a variety of RNA frameworks. Inversely a number of identities can be expressed within a tRNA(Asp) framework. From this emerged the concept of the RNA structural frameworks underlying expression of identities which is illustrated with data obtained with engineered tRNAs. Efficient aspartylation of minihelices is explained by the primordial role of G73. From this and other considerations it is suggested that aspartate identity appeared early in the history of tRNA aminoacylation systems.

Aspartate-tRNA Ligase↗

PubMed1996

A central pseudoknotted three-way junction imposes tRNA-like mimicry and the orientation of three 5' upstream pseudoknots in the 3' terminus of tobacco mosaic virus RNA.

A three-dimensional model of the histidylable 3'-terminal tRNA-like domain of tobacco mosaic virus RNA is proposed on the basis of a comparative structural analysis, chemical and enzymatic probing, combined with graphical modeling of three RNA constructs of increasing size (38, 108, and 182 nt) derived from the 3'-terminal viral RNA sequence. The comparison between the probing patterns of the three RNAs allowed the determination of the relative orientation of these structural domains in the full-length viral tRNA-like structure. Modeling data indicate that only one of the two possible isomers of the three-way junction located at a central position of the tRNA-like domain is in agreement with structural data. Interestingly, this isomer gives rise to a molecule bearing a structural mimicry with the L-shape of canonical tRNAs. A pseudoknotted acceptor branch containing a T-like loop is located perpendicularly to an anticodon-like branch. Moreover, a single-stranded RNA stretch belonging to the pseudoknotted central core mimics a D-like loop and it is proposed that it interacts via two conserved guanosines with nucleotides of the T-like loop as found in canonical tRNAs. This model is valid for the 3' noncoding regions of tobamoviral RNAs as well as for the tRNA-like domain of the satellite tobacco mosaic virus RNA. All three molecules are substrates for yeast HisRS; however, whereas the complete viral genome is required for optimal histidylation capacities, both charging levels and affinity constants are decreased for the three RNA transcripts, suggesting that additional contacts located outside the tRNA-like domain are needed for an optimal aminoacylation process.

Base Sequence↗

PubMed1995

Footprinting of tRNA(Phe) transcripts from Thermus thermophilus HB8 with the homologous phenylalanyl-tRNA synthetase reveals a novel mode of interaction.

The phosphates of the tRNA(Phe) transcript from Thermus thermophilus interacting with the cognate synthetase were determined by footprinting. Backbone bond protection against cleavage by iodine of the phosphorothioate-containing transcripts was found in the anticodon stem-loop, the D stem-loop and the acceptor stem and weak protection was also seen in the variable loop. Most of the protected phosphates correspond to regions around known identity elements of tRNA(Phe). Enhancement of cleavage at certain positions indicates bending of tRNAPhe upon binding to the enzyme. When applied to the three-dimensional model of tRNA(Phe) from yeast the majority of the protections occur on the D loop side of the molecule, revealing that phenylalanyl-tRNA synthetase has a rather complex and novel pattern of interaction with tRNAPhe, differing from that of other known class II aminoacyl-tRNA synthetases.

Base Composition↗

PubMed1995

Cleavage of tRNA with imidazole and spermine imidazole constructs: a new approach for probing RNA structure.

Hydrolysis of RNA in imidazole buffer and by spermine-imidazole conjugates has been investigated. The RNA models were yeast tRNA(Asp) and a transcript derived from the 3'-terminal sequence of tobacco mosaic virus RNA representing a minihelix capable of being enzymatically aminoacylated with histidine. Imidazole buffer and spermine-imidazole conjugates in the presence of free imidazole cleave phosphodiester bonds in the folded RNAs in a specific fashion. Imidazole buffer induces cleavages preferentially in single-stranded regions because nucleotides in these regions have more conformational freedom and can assume more easily the geometry needed for formation of the hydrolysis intermediate state. Spermine-imidazole constructs supplemented with free imidazole cleave tRNA(Asp) within single-stranded regions after pyrimidine residues with a marked preference for pyrimidine-A sequences. Hydrolysis patterns suggest a cleavage mechanism involving an attack by the imidazole residue of the electrostatically bound spermine-imidazole and by free imidazole at the most accessible single-stranded regions of the RNA. Cleavages in a viral RNA fragment recapitulating a tRNA-like domain were found in agreement with the model of this molecule that accounts for its functional properties, thus illustrating the potential of the imidazole-derived reagents as structural probes for solution mapping of RNAs. The cleavage reactions are simple to perform, provide information reflecting the state of the ribose-phosphate backbone of RNA and can be used for mapping single- and double-stranded regions in RNAs.

Base Sequence↗

PubMed1994

Histidylation by yeast HisRS of tRNA or tRNA-like structure relies on residues -1 and 73 but is dependent on the RNA context.

Residue G-1 and discriminator base C73 are the major histidine identity elements in prokaryotes. Here we evaluate the importance of these two nucleotides in yeast histidine aminoacylation identity. Deletion of G-1 in yeast tRNA(His) transcript leads to a drastic loss of histidylation specificity (about 500-fold). Mutation of discriminator base A73, common to all yeast tRNA(His) species, into G73 has a more moderate but still significant effect with a 22-fold decrease in histidylation specificity. Changes at position 36 in the anticodon loop has negligible effect on histidylation. The role of residues -1 and 73 for specific aminoacylation by yeast HisRS was further investigated by studying the histidylation capacities of seven minihelices derived from the Turnip Yellow Mosaic Virus tRNA-like structure. Changes in the nature of nucleotides -1 and 73 modulate this activity but do not suppress it. The optimal mini-substrate for HisRS presents a G.A mismatch at the position equivalent to residues G-1.A73 in yeast tRNA(His), confirms the importance of this structural feature in yeast histidine identity. The fact that the minisubstrates contain a pseudoknot in which position -1 is mimicked by an internal nucleotide from the pseudoknot highlights further the necessity of a stacking interaction of this position over the amino acid accepting branch of the tRNA during the aminoacylation process. Individual transplantation of G-1 or A73 into yeast tRNA(Asp) transcript improves the histidylation efficiency of the engineered tRNA(Asp). However, a tRNA(Asp) transcript presenting simultaneously both residues G-1 and A73 becomes a less good substrate for HisRS, suggesting the importance of the structural context and/or the presence of antideterminants for an optimal expression of these two identity elements.

Anticodon↗

PubMed1994

Identity switches between tRNAs aminoacylated by class I glutaminyl- and class II aspartyl-tRNA synthetases.

High-resolution X-ray structures for the tRNA/aminoacyl-tRNA synthetase complexes between Escherichia coli tRNAGln/GlnRS and yeast tRNAAsp/AspRS have been determined. Positive identity nucleotides that direct aminoacylation specificity have been defined in both cases; E. coli tRNAGln identity is governed by 10 elements scattered in the tRNA structure, while specific aminoacylation of yeast tRNAAsp is dependent on 5 positions. Both identity sets are partially overlapping and share 3 nucleotides. Interestingly, the two enzymes belong to two different classes described for aminoacyl-tRNA synthetases. The class I glutaminyl-tRNA synthetase and the class II aspartyl-tRNA synthetase recognize their cognate tRNA from opposite sides. Mutants derived from glutamine and aspartate tRNAs have been created by progressively introducing identity elements from one tRNA into the other one. Glutaminylation and aspartylation assays of the transplanted tRNAs show that identity nucleotides from a tRNA originally aminoacylated by a synthetase from one class are still recognized if they are presented to the enzyme in a structural framework corresponding to a tRNA aminoacylated by a synthetase belonging to the other class. The simple transplantation of the glutamine identity set into tRNAAsp is sufficient to obtain glutaminylatable tRNA, but additional subtle features seem to be important for the complete conversion of tRNAGln in an aspartylatable substrate. This study defines C38 in yeast tRNAAsp as a new identity nucleotide for aspartylation. We show also in this paper that, during the complex formation, aminoacyl-tRNA synthetases are at least partially responsible for conformational changes which involve structural constraints in tRNA molecules.

Acylation↗

PubMed1994

A histidine accepting tRNA-like fold at the 3'-end of satellite tobacco mosaic virus RNA.

A model of secondary structure is proposed for the 3'-terminal sequence of the satellite tobacco mosaic virus (STMV) RNA on the basis of phylogenetic comparisons with tobacco mosaic virus (TMV) genomic RNA. Sequence homologies and compensatory base changes found between the two related viral RNAs imply that the 3'-end of STMV RNA folds into a tRNA-like domain similar to that found in the TMV RNA. Accordingly, functional assays showed that STMV RNA can be aminoacylated in vitro with histidine by yeast histidyl-tRNA synthetase to plateaus reaching 30%. Histidylation properties of STMV RNA were compared to those of TMV RNA and of a canonical yeast tRNA(His) transcript which both are chargeable to nearly 100% plateau levels. Kinetic data indicate an excellent catalytic efficiency of STMV RNA charging expressed as Vmax/Km ratio, quasi-equivalent to that of TMV RNA, and only 17-fold reduced as compared to that of the yeast tRNAHis transcript. Biological implications of the structural mimicry between the tRNA-like regions of TMV and STMV RNAs are discussed in the light of the relationships of a satellite virus with its helper virus. This is the first report on a chargeable tRNA-like structure at the 3'-end of a satellite virus RNA.

Acylation↗

PubMed1994

Synthetic polyamines stimulate in vitro transcription by T7 RNA polymerase.

The influence of nine synthetic polyamines on in vitro transcription with T7 RNA polymerase has been studied. The compounds used were linear or macrocyclic tetra- and hexaamine, varying in their size, shape and number of protonated groups. Their effect was tested on different types of templates, all presenting the T7 RNA promoter in a double-stranded form followed by sequences encoding short transcripts (25 to 35-mers) either on single- or double-stranded synthetic oligodeoxyribonucleotides. All polyamines used stimulate transcription of both types of templates at levels dependent on their size, shape, protonation degree, and concentration. For each compound, an optimal concentration could be defined; above this concentration, transcription inhibition occurred. Highest stimulation (up to 12-fold) was obtained by the largest cyclic compound called [38]N6C10.

Bacteriophage T7↗

PubMed1994

Crystallogenesis of biological macromolecules: facts and perspectives.

This paper gives an overview of the science of crystals of biological macromolecules. The historical background of the field is outlined and the main achievements and open problems are discussed from both biological and physical-chemical viewpoints. Selected results, including data from the authors, illustrate this overview. The perspectives of crystallogenesis for structural biology, but also more general trends, are presented.

Journal Article↗

PubMed1994

Efficient aminoacylation of resected RNA helices by class II aspartyl-tRNA synthetase dependent on a single nucleotide.

We show here that small RNA helices which recapitulate part or all of the acceptor stem of yeast aspartate tRNA are efficiently aminoacylated by cognate class II aspartyl-tRNA synthetase. Aminoacylation is strongly dependent on the presence of the single-stranded G73 'discriminator' identity nucleotide and is essentially insensitive to the sequence of the helical region. Substrates which contain as few as 3 bp fused to G73CCAOH are aspartylated. Their charging is insensitive to the sequence of the loop closing the short helical domains. Aminoacylation of the aspartate mini-helix is not stimulated by a hairpin helix mimicking the anticodon domain and containing the three major anticodon identity nucleotides. A thermodynamic analysis demonstrates that enzyme interactions with G73 in the resected RNA substrates and in the whole tRNA are the same. Thus, if the resected RNA molecules resemble in some way the earliest substrates for aminoacylation with aspartate, then the contemporary tRNA(Asp) has quantitatively retained the influence of the major signal for aminoacylation in these substrates.

Acylation↗

PubMed1994

Molecular recognition of the identity-determinant set of isoleucine transfer RNA from Escherichia coli.

Molecular recognition of Escherichia coli tRNA(Ile) by the cognate isoleucyl-tRNA synthetase (IleRS) was studied by analyses of chemical footprinting with N-nitroso-N-ethylurea and aminoacylation kinetics of variant tRNA(Ile) transcripts prepared with bacteriophage T7 RNA polymerase. IleRS binds to the acceptor, dihydrouridine (D), and anticodon stems as well as to the anticodon loop. The "complete set" of determinants for the tRNA(Ile) identity consists of most of the nucleotides in the anticodon loop (G34, A35, U36, t6A37 and A38), the discriminator nucleotide (A73), and the base-pairs in the middle of the anticodon, D and acceptor stems (C29.G41, U12.A23 and C4.G69, respectively). As for the tertiary base-pairs, two are indispensable for the isoleucylation activity, whereas the others are dispensable. Correspondingly, some of the phosphate groups of these dispensable tertiary base-pair residues were shown to be exposed to N-nitroso-N-ethylurea when tRNA(Ile) was bound with IleRS. Furthermore, deletion of the T psi C-arm only slightly impaired the tRNA(Ile) activity. Thus, it is proposed that the recognition by IleRS of all the widely distributed identity determinants is coupled with a global conformational change that involves the loosening of a particular set of tertiary base-pairs of tRNA(Ile).

Anticodon↗

PubMed1994

Solution structure of the 3'-end of brome mosaic virus genomic RNAs. Conformational mimicry with canonical tRNAs.

The conformation of the last 201 nucleotides located at the 3'-end of brome mosaic virus (BMV) RNAs was investigated in solution using different chemical and enzymatic probes. Bases were probed with dimethylsulfate (which methylates N-1 positions of A, N-3 positions of C and N-7 positions of G), a carbodiimide (which modifies N-1 positions of G and N-3 positions of U) and diethylpyrocarbonate (which modifies N-7 positions of A). Ribonucleases T1, U2 and S1 were used to map unpaired nucleotides and ribonuclease V1 to monitor paired bases or stacked nucleotides. Cleavage or modification sites were detected by gel electrophoresis either indirectly by analyzing DNA sequence patterns generated by primer extension with reverse transcriptase of the modified RNAs or by direct identification within the statistical cleavage patterns of the RNA. On the basis of these biochemical results, an atomic model was built by computer modeling and its stereochemistry refined. The deduced secondary structure of the RNA confirms data previously proposed by others but contains additional base-pairs (A27-U32, A28-G31, G41-A134, G64-C68, U80-A99, G81-A98, G88-U91, G100-U126, U104-U125, G162-G166 and A172-A191), one new tertiary long-range interaction (U103-U164) and a small triple helical conformation with (G41-A134)-A18 and (C42-G133)-A17 interactions. The new secondary structure also indicates the existence of a second pseudoknot involving pairing between residues A181 to A184 and residues U197 to U194, outside the domain conferring tyrosylation ability to BMV RNA. The main outcome from the model stems from its intricate folding, which allows a new assignment for the domains mimicking the anticodon- and D-loop regions of tRNA. Interestingly, the stem and loop region found structurally to be analogous to the anticodon arm of tRNA(Tyr) does not contain the tyrosine anticodon involved in the aminoacylation process. The structural analogies with canonical tRNA(Tyr) illustrate the functional mimicry existing between the BMV RNA structure and canonical tRNA(Tyr) that allows for their efficient aminoacylation by tyrosyl-tRNA synthetase. This structural model rationalizes mutagenic and footprinting data that have established the importance of specific regions of the viral RNA for recognition by its replicase, (ATP,CTP):tRNA nucleotidyl-transferase and yeast tyrosyl-tRNA synthetase. The new fold has biological implications that can be used as a predictive tool for elaborating new experiments.

Base Sequence↗