PREPARATION AND STRUCTURAL STUDY OF THE DNA-BINDING DOMAIN OF THE CYP1 PROTEIN OF THE YEAST SACCHAROMYCES CEREVISIAE PDF Download
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Author: JOHANNA.. TIMMERMAN
Publisher:
ISBN:
Category :
Languages : fr
Pages : 290
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DANS LA LEVURE SACCHAROMYCES CEREVISIAE, LA PROTEINE CYP1 EST UN REGULATEUR DE LA TRANSCRIPTION DE NOMBREUX GENES DONT L'EXPRESSION EST DEPENDANTE DE L'OXYGENE. NOUS NOUS SOMMES INTERESSE A LA DETERMINATION DE LA STRUCTURE TRIDIMENSIONNELLE DU DOMAINE DE FIXATION A L'ADN DE CETTE PROTEINE. CETTE PARTIE CONTIENT UN MOTIF CYS-XAA(2)-CYS-XAA(6)-CYS-XAA(6)-CYS-XAA(2)-CYS-XAA(8)-CYS, QUE L'ON RETROUVE DANS DE NOMBREUSES PROTEINES DE LEVURE INTERAGISSANT AVEC L'ADN, EN PARTICULIER DANS LA PROTEINE GAL4. LE BUT A LONG TERME EST DE DETERMINER LE COMPLEXE CYP1-ADN PAR RESONANCE MAGNETIQUE NUCLEAIRE. DANS CE TRAVAIL NOUS PRESENTONS LE CLONAGE ET L'EXPRESSION DANS ESCHERICHIA COLI DES QUATRE FRAGMENTS DIFFERENTS CORRESPONDANT A LA PARTIE N-TERMINALE DE L'ACTIVATEUR CYP1 RESPONSABLE DE LA FIXATION A L'ADN. LE FRAGMENT CYP1(49-126) A ETE CHOISI POUR UNE ETUDE STRUCTURALE PAR RESONANCE MAGNETIQUE NUCLEAIRE. POUR CE FRAGMENT, UN PROTOCOLE DE PURIFICATION A ETE MIS AU POINT. NOUS MONTRONS ENSUITE QUE LE ZINC EST UN ELEMENT ESSENTIEL DE LA STRUCTURE DE LA PROTEINE ET QUE LE FRAGMENT CYP1(49-126) POSSEDE 2 ATOMES DE ZINC. LES MEMES RESULTATS ONT ETE OBSERVES LORSQU'ON SUBSTITUE DEUX ATOMES DE ZINC PAR LES ATOMES CADMIUM 113. PAR RESONANCE MAGNETIQUE NUCLEAIRE, NOUS AVONS DEMONTRE QUE LES SIX CYSTEINES DU MOTIF LIGANDENT LES DEUX ATOMES DE CADMIUM. LE FRAGMENT A ETE ENSUITE MARQUE AVEC DE L'AZOTE 15. L'ENSEMBLE DES EXPERIENCES RMN HOMONUCLEAIRES A DEUX DIMENSIONS (COSY, TOCSY ET NOESY) ET HETERONUCLEAIRES A DEUX ET TROIS DIMENSIONS (HMQC, NOE-HMQC, HOHAHA-HMQC) A PERMIS L'ATTRIBUTION DE LA PARTIE N-TERMINALE DE CYP1(49-126). LES DONNEES EXPERIMENTALES OBTENUES PAR CES EXPERIENCES ONT ETE UTILISEES DANS DES PROGRAMMES DE MODELISATION, DIANA ET X-PLOR. UNE STRUCTURE TRIDIMENSIONNELLE DU FRAGMENT A PU ETRE PRESENTEE ET MONTRE QUE NOTRE FRAGMENT EST REPLIE EN CLUSTER A ZINC
Author: JOHANNA.. TIMMERMAN
Publisher:
ISBN:
Category :
Languages : fr
Pages : 290
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Book Description
DANS LA LEVURE SACCHAROMYCES CEREVISIAE, LA PROTEINE CYP1 EST UN REGULATEUR DE LA TRANSCRIPTION DE NOMBREUX GENES DONT L'EXPRESSION EST DEPENDANTE DE L'OXYGENE. NOUS NOUS SOMMES INTERESSE A LA DETERMINATION DE LA STRUCTURE TRIDIMENSIONNELLE DU DOMAINE DE FIXATION A L'ADN DE CETTE PROTEINE. CETTE PARTIE CONTIENT UN MOTIF CYS-XAA(2)-CYS-XAA(6)-CYS-XAA(6)-CYS-XAA(2)-CYS-XAA(8)-CYS, QUE L'ON RETROUVE DANS DE NOMBREUSES PROTEINES DE LEVURE INTERAGISSANT AVEC L'ADN, EN PARTICULIER DANS LA PROTEINE GAL4. LE BUT A LONG TERME EST DE DETERMINER LE COMPLEXE CYP1-ADN PAR RESONANCE MAGNETIQUE NUCLEAIRE. DANS CE TRAVAIL NOUS PRESENTONS LE CLONAGE ET L'EXPRESSION DANS ESCHERICHIA COLI DES QUATRE FRAGMENTS DIFFERENTS CORRESPONDANT A LA PARTIE N-TERMINALE DE L'ACTIVATEUR CYP1 RESPONSABLE DE LA FIXATION A L'ADN. LE FRAGMENT CYP1(49-126) A ETE CHOISI POUR UNE ETUDE STRUCTURALE PAR RESONANCE MAGNETIQUE NUCLEAIRE. POUR CE FRAGMENT, UN PROTOCOLE DE PURIFICATION A ETE MIS AU POINT. NOUS MONTRONS ENSUITE QUE LE ZINC EST UN ELEMENT ESSENTIEL DE LA STRUCTURE DE LA PROTEINE ET QUE LE FRAGMENT CYP1(49-126) POSSEDE 2 ATOMES DE ZINC. LES MEMES RESULTATS ONT ETE OBSERVES LORSQU'ON SUBSTITUE DEUX ATOMES DE ZINC PAR LES ATOMES CADMIUM 113. PAR RESONANCE MAGNETIQUE NUCLEAIRE, NOUS AVONS DEMONTRE QUE LES SIX CYSTEINES DU MOTIF LIGANDENT LES DEUX ATOMES DE CADMIUM. LE FRAGMENT A ETE ENSUITE MARQUE AVEC DE L'AZOTE 15. L'ENSEMBLE DES EXPERIENCES RMN HOMONUCLEAIRES A DEUX DIMENSIONS (COSY, TOCSY ET NOESY) ET HETERONUCLEAIRES A DEUX ET TROIS DIMENSIONS (HMQC, NOE-HMQC, HOHAHA-HMQC) A PERMIS L'ATTRIBUTION DE LA PARTIE N-TERMINALE DE CYP1(49-126). LES DONNEES EXPERIMENTALES OBTENUES PAR CES EXPERIENCES ONT ETE UTILISEES DANS DES PROGRAMMES DE MODELISATION, DIANA ET X-PLOR. UNE STRUCTURE TRIDIMENSIONNELLE DU FRAGMENT A PU ETRE PRESENTEE ET MONTRE QUE NOTRE FRAGMENT EST REPLIE EN CLUSTER A ZINC
Author: Su-Wen Ho
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ISBN:
Category : Electronic dissertations
Languages : en
Pages : 157
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Gene expression is an elaborate and finely tuned process involving the regulated interactions of multiple proteins with promoter and enhancer elements. A variety of approaches are currently used to study these interactions in vivo, in vitro as well as in silico. With the genome sequences of many organisms now readily available, a plethora of DNA functional elements have been predicted, but the process of identifying the proteins that bind to them in vivo remains a bottleneck. I developed two high-throughput assays to address this issue. The first is a modification of the yeast "one-hybrid" assay. The second is probing protein microarrays with DNA sequence elements. Using these methods, I identified two proteins, Sef1 and Yjl103c, that bind to the same DNA sequence element. Sef1 and Yjl103c are little-characterized members of the zinc cluster family of transcription factors of S. cerevisiae. Characterization of their mechanism of action as well as identification of some of their target genes leads to the conclusion that they play a pivotal role in the transcriptional regulation of utilization of nonfermentable carbon sources by budding yeast.
Author: Zhiping Liu
Publisher:
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Category :
Languages : en
Pages : 248
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Author: Anna V. Tchernatynskaia
Publisher:
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Category :
Languages : en
Pages : 414
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In budding yeast, Saccharomyces cerevisiae, a complex of Swi4 and Mbp1 mediates transcriptional activation of many genes during G1-S transition of the cell-cycle. The three-dimensional structure of the consensus binding sequence d(CTTACGCGTCATTG)·d(CAATGACGCGTAAG) has been determined in solution using NMR and rMDs calculation to investigate conformational changes due to complex formation with DNA-binding domain of the Mbp1 protein. 346 distance, 188 torsion angle and residual dipolar coupling restraints were used with Insight II and Xplor-NIH programs to obtain final structures. The Insight and Xplor-NIH structures refined to a mean RMSD 1.46 ± .69Å and 1.26 ± 0.26Å respectively. Both structures belong to the B family of DNA. The global geometry of the structure was significantly improved by incorporation of residual dipolar couplings. The analysis of helical parameters indicates the purine/pyrimidine alternation of the consensus binding site.
![Structure-function Studies of the Yeast Saccharomyces Cerevisiae [alpha]-mating Factor Pheromone Receptor Ste2p PDF](https://books.google.com/books/content/images/frontcover/PtHRjwEACAAJ?fife=w80-h100)
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Category :
Languages : en
Pages :
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G protein-coupled receptors (GPCRs) are seven transmembrane domain cell surface proteins that respond to a variety of environmental cues. Response of these receptors to their cognate stimuli on the extracellular region of the cell results in a concurrent activation of a complex series of intracellular signaling pathways that prepare the cell for the required adjustments through regulation of gene expression levels. Participation of GPCRs in such intricate signal transduction pathways renders them important players in human diseases. The GPCR family of proteins therefore represents one of the largest classes of proteins to be targeted in the development of drug design for clinical applications. In light of the crucial role that GPCRs play in clinically important diseases, the focus of this dissertation has been on interactions between a GPCR and its ligand in a model eukaryotic organism, the budding yeast Saccharomyces cerevisiae. Very recently, the complete genome of the yeast S. cerevisiae has been sequenced. Detailed studies in this system along with the available sequence information have suggested a high conservation between the two eukaryotic organisms human and yeast. Therefore, the S. cerevisiae GPCR Ste2p and its associated pheromone ligand [alpha]-factor represent a good model system to study ligand-receptor interactions. The work presented in this dissertation describes results from a comprehensive mutagenesis approach on Ste2p aimed at determining residues of the receptor that are important in ligand binding and/or receptor activation. Regions of the receptor that have been the primary focus of the studies detailed in this dissertation are the first and third extracellular loops of Ste2p. Additional focus has been given to specific residues located in the transmembrane regions of Ste2p that have been predicted to interact with one another. Cys-scanning and Ala-scanning mutagenesis studies on the first extracellular loop, EL1, of Ste2p resulted in identification of a region of this loop harboring five functionally important residues that played an important role in the activation of the receptor but did not contribute to ligand binding. Structural studies on EL1 pointed to the possibility that this region of EL1 may attain a 310-helical structure in which the five functionally important residues may lie on one face of this helix. Collectively, all these studies underscored the important role of EL1 in Ste2p activation. Structure and function studies on the third extracellular loop, EL3, of Ste2p, using a Cys-scanning mutagenesis approach led to the identification of two additional residues that, upon mutation, resulted in a defective receptor.
Author: R.E. Blankenship
Publisher: Springer Science & Business Media
ISBN: 079233681X
Category : Science
Languages : en
Pages : 1333
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Anoxygenic Photosynthetic Bacteria is a comprehensive volume describing all aspects of non-oxygen-evolving photosynthetic bacteria. The 62 chapters are organized into themes of: Taxonomy, physiology and ecology; Molecular structure of pigments and cofactors; Membrane and cell wall structure: Antenna structure and function; Reaction center structure and electron/proton pathways; Cyclic electron transfer; Metabolic processes; Genetics; Regulation of gene expression, and applications. The chapters have all been written by leading experts and present in detail the current understanding of these versatile microorganisms. The book is intended for use by advanced undergraduate and graduate students and senior researchers in the areas of microbiology, genetics, biochemistry, biophysics and biotechnology.
Author: Munmun S. Nandi
Publisher:
ISBN:
Category :
Languages : en
Pages : 202
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Peroxisomes are virtually found in all eukaryotic cells, but unlike mitochondria and chloroplasts, they do not contain DNA or a protein secretory apparatus. Therefore, all of their proteins must be imported by a process called peroxisomal biogenesis. This requires a group of protein factors referred to as peroxins which are encoded by the pex genes. Currently, there are approximately thirty-two known peroxisomal proteins. Among all the peroxisomal proteins, two enzymes namely GPD1, LYS1 and a peroxin, PEX7 were selected for the research. GPD1 is a NAD+ -dependent glycerol 3-phosphate dehydrogenase1 that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate which is crucial for growth under osmotic stress. Its purification was achieved using ion exchange chromatography and the pure protein was crystallized for structure determination. Diffraction data sets were obtained to a resolution of 2.2 Å which were used to solve the C-terminal portion of the structure. Unfortunately, the N-terminal portion remained disordered. LYS1 is the terminal enzyme of [alpha]-aminoadipate pathway and catalyzes the reversible NAD-dependent oxidative cleavage of saccharopine to yield L-lysine and [alpha]-ketoglutarate. The purification of LYS1 was carried out using affinity chromatography. Another protein, PEX7 is responsible for peroxisome biogenesis by importing newly synthesized proteins bearing PTS2 (peroxisome targeting signal sequence2) into peroxisomes. PEX7 presented the greatest challenge among the three proteins at both the expression stage and the purification stage. Its soluble fraction was purified using ion exchange and affinity chromatographies, although the final yield was too low for crystallization trials. A much large proportion of the protein was found in the insoluble cell debris and attempts were made to purify this fraction after denaturation. An alternative, protocol involving the formation of a GPD1-PEX7 complex proved to be effective route to co-purification of the two proteins and crystallization trials are proceeding. Having known the structures of peroxisomal proteins, it would be helpful for studying the development and maintenance of the organelle related to its metabolic diseases in the eukaryotic cells.
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Category : Biochemistry
Languages : en
Pages : 434
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Category : Medicine
Languages : en
Pages : 1320
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Category : Nucleic acids
Languages : en
Pages : 1158
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