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J C Thierry

Publications and source records attributed to J C Thierry.

At least 19 recordsLinked to original sources
PubMed2003

RASCAL: rapid scanning and correction of multiple sequence alignments.

MOTIVATION: Most multiple sequence alignment programs use heuristics that sometimes introduce errors into the alignment. The most commonly used methods to correct these errors use iterative techniques to maximize an objective function. We present here an alternative, knowledge-based approach that combines a number of recently developed methods into a two-step refinement process. The alignment is divided horizontally and vertically to form a 'lattice' in which well aligned regions can be differentiated. Alignment correction is then restricted to the less reliable regions, leading to a more reliable and efficient refinement strategy. RESULTS: The accuracy and reliability of RASCAL is demonstrated using: (i) alignments from the BAliBASE benchmark database, where significant improvements were often observed, with no deterioration of the existing high-quality regions, (ii) a large scale study involving 946 alignments from the ProDom protein domain database, where alignment quality was increased in 68% of the cases; and (iii) an automatic pipeline to obtain a high-quality alignment of 695 full-length nuclear receptor proteins, which took 11 min on a DEC Alpha 6100 computer AVAILABILITY: RASCAL is available at ftp://ftp-igbmc.u-strasbg.fr/pub/RASCAL. SUPPLEMENTARY INFORMATION: http://bioinfo-igbmc.u-strasbourg.fr/BioInfo/RASCAL/paper/rascal_supp.html

Algorithms↗
PubMed2001

Towards a reliable objective function for multiple sequence alignments.

Multiple sequence alignment is a fundamental tool in a number of different domains in modern molecular biology, including functional and evolutionary studies of a protein family. Multiple alignments also play an essential role in the new integrated systems for genome annotation and analysis. Thus, the development of new multiple alignment scores and statistics is essential, in the spirit of the work dedicated to the evaluation of pairwise sequence alignments for database searching techniques. We present here norMD, a new objective scoring function for multiple sequence alignments. NorMD combines the advantages of the column-scoring techniques with the sensitivity of methods incorporating residue similarity scores. In addition, norMD incorporates ab initio sequence information, such as the number, length and similarity of the sequences to be aligned. The sensitivity and reliability of the norMD objective function is demonstrated using structural alignments in the SCOP and BAliBASE databases. The norMD scores are then applied to the multiple alignments of the complete sequences (MACS) detected by BlastP with E-value<10, for a set of 734 hypothetical proteins encoded by the Vibrio cholerae genome. Unrelated or badly aligned sequences were automatically removed from the MACS, leaving a high-quality multiple alignment which could be reliably exploited in a subsequent functional and/or structural annotation process. After removal of unreliable sequences, 176 (24 %) of the alignments contained at least one sequence with a functional annotation. 103 of these new matches were supported by significant hits to the Interpro domain and motif database.

Amino Acid Motifs↗
PubMed2001

The structure of an AspRS-tRNA(Asp) complex reveals a tRNA-dependent control mechanism.

The 2.6 A resolution crystal structure of an inactive complex between yeast tRNA(Asp) and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit. However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine. The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone- protein interactions. Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism.

Aspartate-tRNA Ligase↗
PubMed2001

BAliBASE (Benchmark Alignment dataBASE): enhancements for repeats, transmembrane sequences and circular permutations.

BAliBASE is specifically designed to serve as an evaluation resource to address all the problems encountered when aligning complete sequences. The database contains high quality, manually constructed multiple sequence alignments together with detailed annotations. The alignments are all based on three-dimensional structural superpositions, with the exception of the transmembrane sequences. The first release provided sets of reference alignments dealing with the problems of high variability, unequal repartition and large N/C-terminal extensions and internal insertions. Here we describe version 2.0 of the database, which incorporates three new reference sets of alignments containing structural repeats, trans-membrane sequences and circular permutations to evaluate the accuracy of detection/prediction and alignment of these complex sequences. BAliBASE can be viewed at the web site http://www-igbmc.u-strasbg. fr/BioInfo/BAliBASE2/index.html or can be downloaded from ftp://ftp-igbmc.u-strasbg.fr/pub/BAliBASE2 /.

Algorithms↗
PubMed2001

Secator: a program for inferring protein subfamilies from phylogenetic trees.

With the huge increase of protein data, an important problem is to estimate, within a large protein family, the number of sensible subsets for subsequent in-depth structural, functional, and evolutionary analyses. To tackle this problem, we developed a new program, Secator, which implements the principle of an ascending hierarchical method using a distance matrix based on a multiple alignment of protein sequences. Dissimilarity values assigned to the nodes of a deduced phylogenetic tree are partitioned by a new stopping rule introduced to automatically determine the significant dissimilarity values. The quality of the clusters obtained by Secator is verified by a separate Jackknife study. The method is demonstrated on 24 large protein families covering a wide spectrum of structural and sequence conservation and its usefulness and accuracy with real biological data is illustrated on two well-studied protein families (the Sm proteins and the nuclear receptors).

Animals↗
PubMed2001

Genome evolution at the genus level: comparison of three complete genomes of hyperthermophilic archaea.

We have compared three complete genomes of closely related hyperthermophilic species of Archaea belonging to the Pyrococcus genus: Pyrococcus abyssi, Pyrococcus horikoshii, and Pyrococcus furiosus. At the genomic level, the comparison reveals a differential conservation among four regions of the Pyrococcus chromosomes correlated with the location of genetic elements mediating DNA reorganization. This discloses the relative contribution of the major mechanisms that promote genomic plasticity in these Archaea, namely rearrangements linked to the replication terminus, insertion sequence-mediated recombinations, and DNA integration within tRNA genes. The combination of these mechanisms leads to a high level of genomic plasticity in these hyperthermophilic Archaea, at least comparable to the plasticity observed between closely related bacteria. At the proteomic level, the comparison of the three Pyrococcus species sheds light on specific selection pressures acting both on their coding capacities and evolutionary rates. Indeed, thanks to two independent methods, the "reciprocal best hits" approach and a new distance ratio analysis, we detect the false orthology relationships within the Pyrococcus lineage. This reveals a high amount of differential gains and losses of genes since the divergence of the three closely related species. The resulting polymorphism is probably linked to an adaptation of these free-living organisms to differential environmental constraints. As a corollary, we delineate the set of orthologous genes shared by the three species, that is, the genes that may characterize the Pyrococcus genus. In this conserved core, the amino acid substitution rate is equal between P. abyssi and P. horikoshii for most of their shared proteins, even for fast-evolving ones. In contrast, strong discrepancies exist among the substitution rates observed in P. furiosus relative to the two other species, which is in disagreement with the molecular clock hypothesis.

Archaeal Proteins↗
PubMed2000

Solution structure of the N-terminal domain of the human TFIIH MAT1 subunit: new insights into the RING finger family.

The human MAT1 protein belongs to the cyclin-dependent kinase-activating kinase complex, which is functionally associated to the transcription/DNA repair factor TFIIH. The N-terminal region of MAT1 consists of a C3HC4 RING finger, which contributes to optimal TFIIH transcriptional activities. We report here the solution structure of the human MAT1 RING finger domain (Met(1)-Asp(65)) as determined by (1)H NMR spectroscopy. The MAT1 RING finger domain presents the expected betaalphabetabeta topology with two interleaved zinc-binding sites conserved among the RING family. However, the presence of an additional helical segment in the N-terminal part of the domain and a conserved hydrophobic central beta strand are the defining features of this new structure and more generally of the MAT1 RING finger subfamily. Comparison of electrostatic surfaces of RING finger structures shows that the RING finger domain of MAT1 presents a remarkable positively charged surface. The functional implications of these MAT1 RING finger features are discussed.

Amino Acid Sequence↗
PubMed2000

Structural characterization of the cysteine-rich domain of TFIIH p44 subunit.

In an effort to understand the structure function relationship of TFIIH, a transcription/repair factor, we focused our attention on the p44 subunit, which plays a central role in both mechanisms. The amino-terminal portion of p44 has been shown to be involved in the regulation of the XPD helicase activity; here we show that its carboxyl-terminal domain is essential for TFIIH transcription activity and that it binds three zinc atoms through two independent modules. The first contains a C4 zinc finger motif, whereas the second is characterized by a CX(2)CX(2-4)FCADCD motif, corresponding to interleaved zinc binding sites. The solution structure of this second module reveals an unexpected homology with the regulatory domain of protein kinase C and provides a framework to study its role at the molecular level.

Amino Acid Sequence↗
PubMed2000

Characterization of crystal content by ESI-MS and MALDI-MS.

A general approach based on mass spectrometry is described for the rapid identification of the content of macromolecular crystals. The experimental procedure was established using lysozyme crystals and then successfully applied to various systems containing specifically bound molecules not easily detectable by other classical techniques. This procedure can be carried out on crystals containing macromolecules of a different nature, such as proteins, nucleic acids and small organic molecules and their non-covalent complexes, grown under various crystallization conditions including PEGs and salts. It can be applied very early on in the crystallization process - as soon as the crystals can be handled. It allows the biologist to control precisely the sequence integrity and homogeneity of the crystallized proteins (in particular at the C-terminus) as well as to verify whether the protein has crystallized with all its expected partners or ligands (nucleic acid molecules, cofactor or small organic molecules).

Aldehyde Reductase↗
PubMed1999

Synthesis of aspartyl-tRNA(Asp) in Escherichia coli--a snapshot of the second step.

The 2.4 A crystal structure of the Escherichia coli aspartyl-tRNA synthetase (AspRS)-tRNA(Asp)-aspartyl-adenylate complex shows the two substrates poised for the transfer of the aspartic acid moiety from the adenylate to the 3'-hydroxyl of the terminal adenosine of the tRNA. A general molecular mechanism is proposed for the second step of the aspartylation reaction that accounts for the observed conformational changes, notably in the active site pocket. The stabilization of the transition state is mediated essentially by two amino acids: the class II invariant arginine of motif 2 and the eubacterial-specific Gln231, which in eukaryotes and archaea is replaced by a structurally non-homologous serine. Two archetypal RNA-protein modes of interactions are observed: the anticodon stem-loop, including the wobble base Q, binds to the N-terminal beta-barrel domain through direct protein-RNA interactions, while the binding of the acceptor stem involves both direct and water-mediated hydrogen bonds in an original recognition scheme.

Anticodon↗
PubMed1998

Crystals of Thermus thermophilus tRNAAsp complexed with its cognate aspartyl-tRNA synthetase have a solvent content of 75%. Comparison with other aminoacylation systems.

Thermus thermophilus tRNAAsp, purified from a non-recombinant source, has been crystallized in a complex with its cognate dimeric (alpha2) aspartyl-tRNA synthetase. Crystals diffract to 2.9 A resolution and belong to space group P63 with cell parameters a = b = 258, c = 90.9 A. The crystals contain one aspartyl-tRNA synthetase dimer and two tRNA molecules in the asymmetric unit, corresponding to a Vm of 4.85 A3 Da-1 and 75% solvent content. When compared with those obtained for globular proteins these values are high, but fall within the range observed for other aminoacyl-tRNA synthetases, either free or complexed with their tRNAs. A comparative survey is presented here.

Amino Acyl-tRNA Synthetases↗
PubMed1998

Crystal structure of aspartyl-tRNA synthetase from Pyrococcus kodakaraensis KOD: archaeon specificity and catalytic mechanism of adenylate formation.

The crystal structure of aspartyl-tRNA synthetase (AspRS) from Pyrococcus kodakaraensis was solved at 1.9 A resolution. The sequence and three-dimensional structure of the catalytic domain are highly homologous to those of eukaryotic AspRSs. In contrast, the N-terminal domain, whose function is to bind the tRNA anticodon, is more similar to that of eubacterial enzymes. Its structure explains the unique property of archaeal AspRSs of accommodating both tRNAAsp and tRNAAsn. Soaking the apo-enzyme crystals with ATP and aspartic acid both separately and together allows the adenylate formation to be followed. Due to the asymmetry of the dimeric enzyme in the crystalline state, different steps of the reaction could be visualized within the same crystal. Four different states of the aspartic acid activation reaction could thus be characterized, revealing the functional correlation of the observed conformational changes. The binding of the amino acid substrate induces movement of two invariant loops which secure the position of the peptidyl moiety for adenylate formation. An unambiguous spatial and functional assignment of three magnesium ion cofactors can be made. This study shows the important role of residues present in both archaeal and eukaryotic AspRSs, but absent from the eubacterial enzymes.

Acylation↗
PubMed1997

The structure of cyclin H: common mode of kinase activation and specific features.

The crystal structure of human cyclin H refined at 2.6 A resolution is compared with that of cyclin A. The core of the molecule consists of two repeats containing five helices each and forming the canonical cyclin fold also observed in TFIIB. One hundred and thirty-two out of the 217 C alpha atoms from the cyclin fold can be superposed with a root-mean-square difference of 1.8 A. The structural homology is even higher for the residues at the interface with the kinase, which is of functional significance, as shown by our observation that cyclin H binds to cyclin-dependent kinase 2 (cdk2) and that cyclin A is able to activate cdk7 in the presence of MAT1. Based on this superposition, a new signature sequence for cyclins was found. The specificity of the cyclin H molecule is provided mainly by two long helices which extend the cyclin fold at its N- and C-termini and pack together against the first repeat on the side opposite to the kinase. Deletion mutants show that the terminal helices are required for a functionally active cyclin H.

Amino Acid Sequence↗
PubMed1997

Expression in Escherichia coli: purification and characterization of cyclin H, a subunit of the human general transcription/DNA repair factor TFIIH.

The human cyclin H, a protein normally associated with the cyclin-dependent kinase cdk7, was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity. The purified recombinant cyclin H is similar to the endogenous one according to the following criteria: molecular weight, microsequencing and mass spectra studies, ability to interact with cdk7, and regulatory kinase activity. The scale-up of cyclin H purification is described.

Affinity Labels↗
PubMed1996

The crystal structure of human cyclin H.

The crystal structure of human cyclin H has been solved at 2.6 A resolution by the MIR method and refined to an R-factor of 23.1%. The core of the molecule consists of two helical repeats adopting the canonical cyclin fold already observed in the structures of cyclin A [Brown et al. (1995) Structure 3, 1235-1247; Jeffrey et al. (1995) Nature 376, 313-320; Russo et al. (1996) Nature 382, 325-331] and TFIIB [Nikoilov et al. (1995) Nature 377, 119-128]. The N-terminal and C-terminal residues form a new domain built on two long helices interacting essentially with the first repeat of the molecule.

Crystallization↗
PubMed1995

The class II aminoacyl-tRNA synthetases and their active site: evolutionary conservation of an ATP binding site.

Previous sequence analyses have suggested the existence of two distinct classes of aminoacyl-tRNA synthetase. The partition was established on the basis of exclusive sets of sequence motifs (Eriani et al. [1990] Nature 347:203-306). X-ray studies have now well defined the structural basis of the two classes: the class I enzymes share with dehydrogenases and kinases the classic nucleotide binding fold called the Rossmann fold, whereas the class II enzymes possess a different fold, not found elsewhere, built around a six-stranded antiparallel beta-sheet. The two classes of synthetases catalyze the same global reaction that is the attachment of an amino acid to the tRNA, but differ as to where on the terminal adenosine of the tRNA the amino acid is placed: class I enzymes act on the 2' hydroxyl whereas the class II enzymes prefer the 3' hydroxyl group. The three-dimensional structure of aspartyl-tRNA synthetase from yeast, a typical class II enzyme, is described here, in relation to its function. The crucial role of the sequence motifs in substrate binding and enzyme structure is high-lighted. Overall these results underline the existence of an intimate evolutionary link between the aminoacyl-tRNA synthetases, despite their actual structural diversity.

Adenosine Triphosphate↗