Yeast Structural Genomics

Analysis and prediction of protein quaternary structure

Anne — 2008-04-26T11:10:20Z

Abstract

The quaternary structure (QS) of a protein is determined by measuring its molecular weight in solution. The data have to be extracted from the literature, and they may be missing even for proteins that have a crystal structure reported in the Protein Data Bank (PDB). The PDB and other databases derived from it, report QS information that either was obtained from the depositors, or is based on an analysis of the contacts between polypeptide chains in the crystal, and this frequently differs from the QS determined in solution. The QS of a protein can be predicted from its sequence using either homology or threading methods. However, a majority of the proteins with less than 30% sequence identity have different QS. A model of the QS can also be derived by docking the subunits when their 3D structure is independently known, but the model is likely to be incorrect if large conformation changes take place when the oligomer assembles.

Mesures Hi�rarchiques pond�r�es pour l'�valuation d' un syst�me semi-automatique d'annotation de g�nomes utilisant des arbres de d�cision

Anne — 2008-01-31T17:49:01Z

8�mes journ�es francophones "Extraction et Gestion des Connaissances"

R�sum�

Dans le cadre de l'annotation fonctionnelle de prot�ines, l'attribution d'une fonction se fait, entre autres, � l'aide d'une hi�rarchie fonctionnelle. Celle-ci permet d'organiser les connaissances biologiques et d'utiliser un vocabulaire contr�l� sous la forme d'une hi�rarchie. Pour estimer la pertinence des annotations, des mesures telles que la pr�cision, le rappel, la sp�cificit�, le Fscore sont utilis�es. Cependant ces mesures ne sont pas toujours bien adapt�es � l'�valuation de donn�es hi�rarchiques. Nous proposons, ici, plusieurs mesures plus appropri�es � notre probl�me.

The VIZIER project: Preparedness against pathogenic RNA viruses.

Anne — 2008-01-31T16:33:44Z

Abstract

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

DiMoVo: a Voronoi tessellation-based method for discriminating crystallographic and biological protein-protein interactions.

Anne — 2008-01-31T16:32:31Z

Abstract

MOTIVATION: Knowledge of the oligomeric state of a protein is often essential for understanding its function and mechanism. Within a protein crystal, each protein monomer is in contact with many others, forming many small interfaces and a few larger ones that are biologically significant if the protein is a homodimer in solution, but not if the protein is monomeric. Telling such "crystal dimers" from real ones remains a difficult task. RESULTS: It has already been demonstrated that the interfaces of native and non-native protein-protein complexes can be distinguished using a combination of parameters computed with a method on the Voronoi tessellation (Bernauer, et al., 2007). We show in this paper that the same parameters highlight significant differences between the interfaces of biological and crystal dimers. Using these parameters as descriptors in machine learning methods leads to accurate classification of specific and non-specific protein-protein interfaces. AVAILABILITY: http://fifi.ibbmc.u-psud.fr/DiMoVo.

The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases.

Anne — 2008-01-31T16:29:50Z

Abstract

Emg1 was previously shown to be required for maturation of the 18S rRNA and biogenesis of the 40S ribosomal subunit. Here we report the determination of the crystal structure of Emg1 at 2 A resolution in complex with the methyl donor, S-adenosyl-methionine (SAM). This structure identifies Emg1 as a novel member of the alpha/beta knot fold methyltransferase (SPOUT) superfamily. In addition to the conserved SPOUT core, Emg1 has two unique domains that form an extended surface, which we predict to be involved in binding of RNA substrates. A point mutation within a basic patch on this surface almost completely abolished RNA binding in vitro. Three point mutations designed to disrupt the interaction of Emg1 with SAM each caused>100-fold reduction in SAM binding in vitro. Expression of only Emg1 with these mutations could support growth and apparently normal ribosome biogenesis in strains genetically depleted of Emg1. We conclude that the catalytic activity of Emg1 is not essential and that the presence of the protein is both necessary and sufficient for ribosome biogenesis.

Structure of yeast Dom34: a protein related to translation termination factor eRF1 and involved in No-Go decay.

Anne — 2008-01-31T16:28:49Z

Abstract

The yeast protein Dom34 has been described to play a critical role in a newly identified mRNA decay pathway called No-Go decay. This pathway clears cells from mRNAs inducing translational stalls through endonucleolytic cleavage. Dom34 is related to the translation termination factor eRF1 and physically interacts with Hbs1, which is itself related to eRF3. We have solved the 2.5A resolution crystal structure of S. cerevisiae Dom34. This protein is organized in three domains with the central and C-terminal domains structurally homologous to those from eRF1. The N-terminal domain of Dom34 is different from eRF1. It adopts a Sm-fold which is often involved in the recognition of mRNA stem loops or in the recruitment of mRNA degradation machinery. The comparison of eRF1 and Dom34 domains proposed to interact directly with eRF3 and Hbs1, respectively, highlights striking structural similarities with eRF1 motifs identified to be crucial for the binding to eRF3. In addition, as observed for eRF1 which enhances eRF3 binding to GTP, the interaction of Dom34 with Hbs1 results in an increase in the affinity constant of Hbs1 for GTP but not GDP. Taken together, these results emphasize that eukaryotic cells have evolved two structurally related complexes able to interact with ribosomes either paused at a stop codon or stalled in translation by the presence of a stable stem-loop and to trigger ribosome release by catalyzing chemical bond hydrolysis.

Structure of the Yeast tRNA m7G Methylation Complex

Anne — 2008-01-31T16:27:12Z

Abstract

Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNA(Phe) has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.

Towards a semi-automatic functional annotation tool based on decision tree techniques

Anne — 2007-09-29T09:25:55Z

Due to the continuous improvements of high throughput technologies and experimental procedures, the number of sequenced genomes is increasing exponentially. Biologist experts play a central role in the analysis of this massive amount of raw data. To annotate a new genome they need to integrate many pieces of information coming from various sources: results of bioinformatics analysis programs, data stored in specialized databases, results of high-throughput experiments such as transcriptomics, proteomics, etc., information stored in the literature, general knowledge about the domain of interest (biological properties of the studied organism, its ecology, etc.). To face the deluge of new genomic data, there is a crying need to automate, as far as possible, the annotation process itself.

The rolling-circle plasmid pTN1 from the hyperthermophilic archaeon Thermococcus nautilus.

Anne — 2007-09-29T09:14:27Z

The hyperthermophilic archaeon Thermococcus nautilus carries a plasmid, pTN1, which encodes a rolling-circle (RC) replication initiator protein of 74 kDa (Rep74) and an orphan protein of 24 kDa (p24). The Rep74 protein is homologous to the Rep75 protein encoded by the RC plasmid pGT5 from Pyrococcus abyssi. Comparative analysis of Rep74 and Rep75 sequences shows that these proteins correspond to a new family of RC initiators formed by the fusion of a Rep domain with an N-terminal domain of unknown function. Surprisingly, the Rep domain of Rep74/75 is more closely related to transposases encoded by IS elements than to Rep proteins of other RC plasmids. The p24 protein contains a hydrophobic segment, a highly charged region and a zinc finger motif. A recombinant p24 protein lacking the hydrophobic segment binds and condenses both single- and double-stranded DNA, and forms DNA aggregates with extreme compaction at high protein to DNA ratio. In addition to encoding proteins of significant interest, pTN1 is remarkable by being the only characterized plasmid isolated from a Thermococcus strain, thus being useful to develop genetic tools in Thermococcus kodakaraensis for which gene disruption methods became recently available.

An archaeal orthologue of the universal protein Kae1 is an iron metalloprotein which exhibits atypical DNA-binding properties and apurinic-endonuclease activity in vitro.

Anne — 2007-09-29T09:12:15Z

The Kae1 (Kinase-associated endopeptidase 1) protein is a member of the recently identified transcription complex EKC and telomeres maintenance complex KEOPS in yeast. Kae1 homologues are encoded by all sequenced genomes in the three domains of life. Although annotated as putative endopeptidases, the actual functions of these universal proteins are unknown. Here we show that the purified Kae1 protein (Pa-Kae1) from Pyrococcus abyssi is an iron-protein with a novel type of ATP-binding site. Surprisingly, this protein did not exhibit endopeptidase activity in vitro but binds cooperatively to single and double-stranded DNA and induces unusual DNA conformational change. Furthermore, Pa-Kae1 exhibits a class I apurinic (AP)-endonuclease activity (AP-lyase). Both DNA binding and AP-endonuclease activity are inhibited by ATP. Kae1 is thus a novel and atypical universal DNA interacting protein whose importance could rival those of RecA (RadA/Rad51) in the maintenance of genome integrity in all living cells.