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Artykuły w czasopismach na temat "Zinc finger motifs"
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Zhang, XiaoHong, YuJi Miao, XiaoDan Hu, Rui Min, PeiDang Liu i HaiQian Zhang. "Gamma Radiation-Induced Damage in the Zinc Finger of the Transcription Factor IIIA". Bioinorganic Chemistry and Applications 2016 (2016): 1–7. http://dx.doi.org/10.1155/2016/1642064.
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A zinc finger motif is an element of proteins that can specifically recognize and bind to DNA. Because they contain multiple cysteine residues, zinc finger motifs possess redox properties. Ionizing radiation generates a variety of free radicals in organisms. Zinc finger motifs, therefore, may be a target of ionizing radiation. The effect of gamma radiation on the zinc finger motifs in transcription factor IIIA (TFIIIA), a zinc finger protein, was investigated. TFIIIA was exposed to different gamma doses from 60Co sources. The dose rates were 0.20 Gy/min and 800 Gy/h, respectively. The binding capacity of zinc finger motifs in TFIIIA was determined using an electrophoretic mobility shift assay. We found that 1000 Gy of gamma radiation impaired the function of the zinc finger motifs in TFIIIA. The sites of radiation-induced damage in the zinc finger were the thiol groups of cysteine residues and zinc (II) ions. The thiol groups were oxidized to form disulfide bonds and the zinc (II) ions were indicated to be reduced to zinc atoms. These results indicate that the zinc finger motif is a target domain for gamma radiation, which may decrease 5S rRNA expression via impairment of the zinc finger motifs in TFIIIA.
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GREEN, Andrew, i Bibudhendra SARKAR. "Alteration of zif268 zinc-finger motifs gives rise to non-native zinc-co-ordination sites but preserves wild-type DNA recognition". Biochemical Journal 333, nr 1 (1.07.1998): 85–90. http://dx.doi.org/10.1042/bj3330085.
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Zinc fingers are among the major structural motifs found in proteins that are involved in eukaryotic gene regulation. Many of these zinc-finger domains are involved in DNA binding. This study investigated whether the zinc-co-ordinating (Cys)2(His)2 motif found in the three zinc fingers of zif268 could be replaced by a (Cys)4 motif while still preserving DNA recognition. (Cys)2(His)2-to-(Cys)4 mutations were generated in each of the three zinc fingers of zif268 individually, as well as in fingers 1 and 3, and fingers 2 and 3 together. Whereas finger 1 and finger 3 tolerate the switch, such an alteration in finger 2 renders the polypeptide incapable of DNA recognition. The protein–DNA interaction was examined in greater detail by using a methylation-interference assay. The mutant polypeptides containing the (Cys)4 motif in fingers 1 or 3 recognize DNA in a manner identical to the wild-type protein, suggesting that the (Cys)4 motif appears to give rise to a properly folded finger. Additional results indicate that a zif268 variant containing a (Cys)2(His)(Ala) arrangement in finger 1 is also capable of DNA recognition in a manner identical to the wild-type polypeptide. This appears to be the first time that such alterations, in the context of an intact DNA-binding domain, have still allowed for specific DNA recognition. Taken together, the work presented here enhances our understanding of the relationship between metal ligation and DNA-binding by zinc fingers.
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MAURER-STROH, SEBASTIAN, HE GAO, HAO HAN, LIES BAETEN, JOOST SCHYMKOWITZ, FREDERIC ROUSSEAU, LOUXIN ZHANG i FRANK EISENHABER. "MOTIF DISCOVERY WITH DATA MINING IN 3D PROTEIN STRUCTURE DATABASES: DISCOVERY, VALIDATION AND PREDICTION OF THE U-SHAPE ZINC BINDING ("HUF-ZINC") MOTIF". Journal of Bioinformatics and Computational Biology 11, nr 01 (luty 2013): 1340008. http://dx.doi.org/10.1142/s0219720013400088.
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Data mining in protein databases, derivatives from more fundamental protein 3D structure and sequence databases, has considerable unearthed potential for the discovery of sequence motif—structural motif—function relationships as the finding of the U-shape (Huf-Zinc) motif, originally a small student's project, exemplifies. The metal ion zinc is critically involved in universal biological processes, ranging from protein-DNA complexes and transcription regulation to enzymatic catalysis and metabolic pathways. Proteins have evolved a series of motifs to specifically recognize and bind zinc ions. Many of these, so called zinc fingers, are structurally independent globular domains with discontinuous binding motifs made up of residues mostly far apart in sequence. Through a systematic approach starting from the BRIX structure fragment database, we discovered that there exists another predictable subset of zinc-binding motifs that not only have a conserved continuous sequence pattern but also share a characteristic local conformation, despite being included in totally different overall folds. While this does not allow general prediction of all Zn binding motifs, a HMM-based web server, Huf-Zinc, is available for prediction of these novel, as well as conventional, zinc finger motifs in protein sequences. The Huf-Zinc webserver can be freely accessed through this URL ( http://mendel.bii.a-star.edu.sg/METHODS/hufzinc/ ).
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Gebelein, Brian, i Raul Urrutia. "Sequence-Specific Transcriptional Repression by KS1, a Multiple-Zinc-Finger–Krüppel-Associated Box Protein". Molecular and Cellular Biology 21, nr 3 (1.02.2001): 928–39. http://dx.doi.org/10.1128/mcb.21.3.928-939.2001.
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ABSTRACT The vertebrate genome contains a large number of Krüppel-associated box–zinc finger genes that encode 10 or more C2-H2 zinc finger motifs. Members of this gene family have been proposed to function as transcription factors by binding DNA through their zinc finger region and repressing gene expression via the KRAB domain. To date, however, no Krüppel-associated box–zinc finger protein (KRAB-ZFP) and few proteins with 10 or more zinc finger motifs have been shown to bind DNA in a sequence-specific manner. Our laboratory has recently identified KS1, a member of the KRAB-ZFP family that contains 10 different C2-H2 zinc finger motifs, 9 clustered at the C terminus with an additional zinc finger separated by a short linker region. In this study, we used a random oligonucleotide binding assay to identify a 27-bp KS1 binding element (KBE). Reporter assays demonstrate that KS1 represses the expression of promoters containing this DNA sequence. Deletion and site-directed mutagenesis reveal that KS1 requires nine C-terminal zinc fingers and the KRAB domain for transcriptional repression through the KBE site, whereas the isolated zinc finger and linker region are dispensable for this function. Additional biochemical assays demonstrate that the KS1 KRAB domain interacts with the KAP-1 corepressor, and mutations that abolish this interaction alleviate KS1-mediated transcriptional repression. Thus, this study provides the first direct evidence that a KRAB-ZFP binds DNA to regulate gene expression and provides insight into the mechanisms used by multiple-zinc-finger proteins to recognize DNA sequences.
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Hasegawa, Atsushi, Hiroshi Kaneko, Daishi Ishihara, Masahiro Nakamura, Akira Watanabe, Cecelia D. Trainor, Yamamoto Masayuki i Ritsuko Shimizu. "GATA1 Changes DNA-Binding Fashion in a Binding-Site-Specific Manner and Alters Transcriptional Activity during Erythropoiesis". Blood 126, nr 23 (3.12.2015): 3584. http://dx.doi.org/10.1182/blood.v126.23.3584.3584.
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Abstract GATA1 is a transcription factor that coordinately regulates multiple target genes during the development and differentiation of erythroid and megakaryocytic lineages through binding to GATA motif (A/T)GATA(A/G). GATA1 has four functional domains, i.e., two transactivation domains reside in amino- and carboxyl- terminus, which transactivate GATA1 target genes redundantly and/or cooperatively, and two zinc-finger domains in the middle of the protein. The two zinc finger domains of GATA1 have been characterized extensively and their links to human diseases have also been identified. Carboxyl-terminal side zinc (C)-finger is essential for the DNA binding of GATA1, whereas amino-terminal side zinc (N)-finger retains insufficient binding activity to the GATA motifs by itself, but contributes to stabilize the binding of C-finger to a double GATA site arranged in a palindromic manner. Of note, while this two-finger structure is conserved in six distinct vertebrate GATA factors, there exist GATA factors with single zinc finger in non-vertebrates, indicating that only the C-finger and following basic tail region are evolutionary conserved in both vertebrate and non-vertebrate GATA factors. In our transgenic rescue analyses, GATA1 lacking the N-finger (ΔNF-GATA1) supports, if not completely, the erythropoiesis in mice, but mice without C-finger (ΔCF-GATA1) die in utero showing similar phenotype to the mice with complete loss-of-GATA1-function. Therefore, roles that the N-finger plays have been assumed to be evolutionally acquired features during molecular evolution. In this study, we have examined GATA-motif configuration-specific modulation of GATA1 function by using composite GATA elements in which two GATA motifs aligned side-by-side, either tandem or palindromic. We have defined changes in the GATA1 binding and transactivation activity in accordance with the arrangement of cis -acting GATA motifs. While GATA1 binds to Single-GATA in a monovalent way via C-finger without the influence of N-finger, the N-finger appears to contribute to specific bivalent binding of GATA1 to Pal-GATA, i.e., the N- and C-fingers in a single GATA1 molecule individually bind to two GATA motifs aligned in a palindromic orientation. Showing very good agreement with the human case analyses, the transgenic expression of G1R216Q that lacks N-finger-DNA interaction potential hardly rescues the GATA1-deficient mice due to defects in definitive erythropoiesis, indicating that roles owed by R216 residue are vital for the GATA1 activity in vivo. The N-finger also contributes to GATA1 homodimer formation, which is a prerequisite for two GATA1 binding to two GATA motifs aligned in a tandem orientation. Each GATA1 C-finger in the dimeric GATA1 protein binds to each GATA motif in Tandem-GATA. In this regard, we previously found in a transgenic complementation rescue assay that mutant GATA1 molecule G13KA, which lacks the dimerization potential but possesses most of the other N- and C-finger functions, hardly rescues the GATA1-deficient mice from embryonic lethality, indicating that the GATA1 dimerization is important to attain full GATA1 activity. We surmise based on these observations that the configuration of cis -acting GATA motifs located in the regulatory regions of the GATA1 target genes critically influences the DNA-binding of GATA1 and controls transcription of the genes. Disclosures No relevant conflicts of interest to declare.
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Parraga, G., L. Young i R. E. Klevit. "Zinc-finger motifs and DNA binding". Trends in Biochemical Sciences 14, nr 10 (październik 1989): 398. http://dx.doi.org/10.1016/0968-0004(89)90283-1.
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Gao, Xiang, Daniel J. Rowley, Xiaowu Gai i Daniel F. Voytas. "Ty5 gag Mutations Increase Retrotransposition and Suggest a Role for Hydrogen Bonding in the Function of the Nucleocapsid Zinc Finger". Journal of Virology 76, nr 7 (1.04.2002): 3240–47. http://dx.doi.org/10.1128/jvi.76.7.3240-3247.2002.
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ABSTRACT The Ty5 retrotransposon of Saccharomyces paradoxus transposes in Saccharomyces cerevisiae at frequencies 1,000-fold lower than do the native Ty1 elements. The low transposition activity of Ty5 could be due to differences in cellular environments between these yeast species or to naturally occurring mutations in Ty5. By screening of a Ty5 mutant library, two single mutants (D252N and Y68C) were each found to increase transposition approximately sixfold. When combined, transposition increased 36-fold, implying that the two mutations act independently. Neither mutation affected Ty5 protein synthesis, processing, cDNA recombination, or target site choice. However, cDNA levels in both single mutants and the double mutant were significantly higher than in the wild type. The D252N mutation resides in the zinc finger of nucleocapsid and increases the potential for hydrogen bonding with nucleic acids. We generated other mutations that increase the hydrogen bonding potential (i.e., D252R and D252K) and found that they similarly increased transposition. This suggests that hydrogen bonding within the zinc finger motif is important for cDNA production and builds upon previous studies implicating basic amino acids flanking the zinc finger as important for zinc finger function. Although NCp zinc fingers differ from the zinc finger motifs of cellular enzymes, the requirement for efficient hydrogen bonding is likely universal.
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Bowzard, J. Bradford, Robert P. Bennett, Neel K. Krishna, Sandra M. Ernst, Alan Rein i John W. Wills. "Importance of Basic Residues in the Nucleocapsid Sequence for Retrovirus Gag Assembly and Complementation Rescue". Journal of Virology 72, nr 11 (1.11.1998): 9034–44. http://dx.doi.org/10.1128/jvi.72.11.9034-9044.1998.
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ABSTRACT The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) contain small interaction (I) domains within their nucleocapsid (NC) sequences. These overlap the zinc finger motifs and function to provide the proper density to viral particles. There are two zinc fingers and at least two I domains within these Gag proteins. To more thoroughly characterize the important sequence features and properties of I domains, we analyzed Gag proteins that contain one or no zinc finger motifs. Chimeric proteins containing the amino-terminal half of RSV Gag and various portions of the carboxy terminus of murine leukemia virus (MLV) (containing one zinc finger) Gag had only one I domain, whereas similar chimeras with human foamy virus (HFV) (containing no zinc fingers) Gag had at least two. Mutational analysis of the MLV NC sequence and inspection of I domain sequences within the zinc-fingerless C terminus of HFV Gag suggested that clusters of basic residues, but not the zinc finger motif residues themselves, are required for the formation of particles of proper density. In support of this, a simple string of strongly basic residues was found to be able to substitute for the RSV I domains. We also explored the possibility that differences in I domains (e.g., their number) account for differences in the ability of Gag proteins to be rescued into particles when they are unable to bind to membranes. Previously published experiments have shown that such membrane-binding mutants of RSV and HIV (two I domains) can be rescued but that those of MLV (one I domain) cannot. Complementation rescue experiments with RSV-MLV chimeras now map this difference to the NC sequence of MLV. Importantly, the same RSV-MLV chimeras could be rescued by complementation when the block to budding was after, rather than before, transport to the membrane. These results suggest that MLV Gag molecules begin to interact at a much later time after synthesis than those of RSV and HIV.
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Guo, Jianhui, Tiyun Wu, Bradley F. Kane, Donald G. Johnson, Louis E. Henderson, Robert J. Gorelick i Judith G. Levin. "Subtle Alterations of the Native Zinc Finger Structures Have Dramatic Effects on the Nucleic Acid Chaperone Activity of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein". Journal of Virology 76, nr 9 (1.05.2002): 4370–78. http://dx.doi.org/10.1128/jvi.76.9.4370-4378.2002.
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ABSTRACT The nucleocapsid protein (NC) of human immunodeficiency virus type 1 has two zinc fingers, each containing the invariant CCHC zinc-binding motif; however, the surrounding amino acid context is not identical in the two fingers. Recently, we demonstrated that zinc coordination is required when NC unfolds complex secondary structures in RNA and DNA minus- and plus-strand transfer intermediates; this property of NC reflects its nucleic acid chaperone activity. Here we have analyzed the chaperone activities of mutants having substitutions of alternative zinc-coordinating residues, i.e., CCHH or CCCC, for the wild-type CCHC motif. We also investigated the activities of mutants that retain the CCHC motifs but have mutations that exchange or duplicate the zinc fingers (mutants 1-1, 2-1, and 2-2); these changes affect amino acid context. Our results indicate that in general, for optimal activity in an assay that measures stimulation of minus-strand transfer and inhibition of nonspecific self-priming, the CCHC motif in the zinc fingers cannot be replaced by CCHH or CCCC and the amino acid context of the fingers must be conserved. Context changes also reduce the ability of NC to facilitate primer removal in plus-strand transfer. In addition, we found that the first finger is a more crucial determinant of nucleic acid chaperone activity than the second finger. Interestingly, comparison of the in vitro results with earlier in vivo replication data raises the possibility that NC may adopt multiple conformations that are responsible for different NC functions during virus replication.
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Guo, Xuemin, John-William N. Carroll, Margaret R. MacDonald, Stephen P. Goff i Guangxia Gao. "The Zinc Finger Antiviral Protein Directly Binds to Specific Viral mRNAs through the CCCH Zinc Finger Motifs". Journal of Virology 78, nr 23 (1.12.2004): 12781–87. http://dx.doi.org/10.1128/jvi.78.23.12781-12787.2004.
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ABSTRACT The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. For this report, we mapped the viral sequences that are sensitive to ZAP inhibition. The viral sequences were cloned into a luciferase reporter and analyzed for the ability to mediate ZAP-dependent destabilization of the reporter. The sensitive sequence in MLV was mapped to the 3′ long terminal repeat; the sensitive sequences in SIN were mapped to multiple fragments. The fragment of SIN that displayed the highest destabilizing activity was further analyzed by deletion mutagenesis for the minimal sequence that retained the activity. This led to the identification of a fragment of 653 nucleotides. Any further deletion of this fragment resulted in significantly lower activity. We provide evidence that ZAP directly binds to the active but not the inactive fragments. The CCCH zinc finger motifs of ZAP play important roles in RNA binding and antiviral activity. Disruption of the second and fourth zinc fingers abolished ZAP's activity, whereas disruption of the first and third fingers just slightly lowered its activity.
Rozprawy doktorskie na temat "Zinc finger motifs"
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Wang, Zhonghua Laity John H. "Characterization of novel structure-regulatory relationships within interacting two-finger Cys₂His₂ zinc finger protein motifs". Diss., UMK access, 2008.
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Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2008."A dissertation in cell biology and biophysics and molecular biology and biochemistry." Advisor: John H. Laity. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Sept.12, 2008. Includes bibliographical references (leaves 148-166). Online version of the print edition.
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Guéguéniat, Julia. "Étude fonctionnelle des sous-domaines de Pcf11 : rôle du 2nd NTD dans la terminaison de transcription des snoRNAs et des motifs liant le zinc dans les activités de maturation de l’extrémité 3’ des ARN messagers". Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0236/document.
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Chez les eucaryotes, la maturation de l’extrémité 3’ des ARNs messagers a lieu lors de la transcription et regroupe deux étapes : le clivage endonucléolytique du transcrit au niveau d’un site spécifique et l’ajout d’une queue poly(A) sur le fragment en amont du site de clivage. Chez S. cerevisiae, le complexe de polyadénylation est formé par 20 protéines, regroupées principalement en deux sous-complexes : CF IA et CPF. Nous nous intéressons plus spécifiquement à Pcf11, sous-unité du complexe CF IA. Pcf11 est formé de sept sous-domaines, mais la fonction d’une grande partie de la protéine n’est pour l’instant pas connue. Par exemple, aucune fonction n’est associée à la région située entre le domaine d’interaction avec le CTD de l’ARN polymérase II (CID) et une répétion de 20 résidus glutamines. Récemment, la structure de ce domaine, appelé 2nd NTD a été décrite. Pour essayer de comprendre la fonction du 2nd NTD et des motifs liant le zinc encadrant le domaine d’interaction avec Clp1, nous avons mis en place une stratégie systématique de mutagénèse, soit par délétions, soit par mutations ponctuelles. Le 2nd NTD est formé de trois hélices α et interagit avec l’ARN. La délétion de ce domaine conduit à un phénotype de croissance lente chez la levure et un défaut de terminaison de transcription des snoRNAs. Malgré une similarité de structure et de fonction, le 2nd NTD présenterait une fonction indépendante. La fonction des motifs liant le zinc n’est pour l’instant pas connue. Cependant, la mutation de l’un de ces deux motifs conduit à un défaut de clivage et de polyadénylation in vitro. La mutation des deux motifs est létale chez la levureIn eukaryotes, poly (A) tails are added to nuclear pre-mRNA 3'-ends in the two steps of cleavage and polyadenylation. This co-transcriptional processing requires the activity of a large protein complex comprising at least 20 different polypeptides in yeast organized primarily into the two factors CF IA and CPF. We are interested in the functional characterization of Pcf11, a CF IA subunit. The Pcf11 protein is organized into seven different domains, but here is still a large portion of the polypeptide that has not yet been characterized. For example the region from the end of the CTD interaction domain (CID) to an uninterrupted stretch of 20 glutamine residues has no known function. Recently, the structure of this region, called the 2nd NTD have been characterized. To gain insight into the function of the 2nd NTD and the two zinc fingers motif surrounding the Clp1 interaction domain, we have employed a systematic strategy of mutagenesis, either by deletion or via point mutations. The 2nd NTD is a folded domain composed of three α-helices. The deletion of this domain induced a severe defect of growth in yeast and impaired transcription termination of snoRNAs. Despite its similarity in structure and function with the CID, the 2nd NTD seems to act like an independent RNA binding domain. We don’t know yet the real function of the two zinc fingers motif at the C-terminal region of Pcf11, but the mutation of Cystein residues into serine of one of the two motifs impaired cleavage and polyadenylation. The mutation of the first motif is less harmful than the mutation of the second motif. The simultaneous mutation is lethal in yeast
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Oliveira, Alessandra Rejane Ericsson de. "Identificação e caracterização de uma proteína com motivos ZINC FINGER de Trypanosoma cruzi". reponame:Repositório Institucional da UnB, 2006. http://repositorio.unb.br/handle/10482/3320.
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Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2006.Submitted by Larissa Ferreira dos Angelos (ferreirangelos@gmail.com) on 2009-11-14T12:47:05Z No. of bitstreams: 1 2006_Alessandra Rejane Ericsson de Oliveira.pdf: 1765643 bytes, checksum: 9a175047678704e91e49247033e86e47 (MD5)
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Proteínas zinc finger são compostas por domínios compactados contendo α- hélices e folhas β unidos e estabilizados por um ou dois átomos de zinco. Arranjos repetidos de zinc fingers são comumente utilizados para reconhecimento de ácidos nucléicos. Dentre outras atividades, eles estão envolvidos em replicação, transcrição e reparo de DNA. No nucleocapsídeo do vírus HIV tipo 1 foi identificada uma proteína contendo o motivo zinc finger CX2CX4HX4C, estando esta proteína envolvida em várias etapas do ciclo de vida viral, incluindo a propriedade de encapsidação do RNA viral. Em tripanosomatídeos, somente poucas proteínas contendo o motivo zinc finger já foram identificadas até o presente momento. Em um fragmento genômico de 17 kb da banda XX de T. cruzi, nós identificamos três genes in tandem codificando para proteínas zinc finger do tipo CX2CX2HX4C. Nós também demonstramos que genes similares estão presentes em T. brucei e L. major como três definidos grupos monofiléticos entre esses tripanosomatídeos. Em T. cruzi, TcZFP8 corresponde a um novo gene codificando para uma proteína com oito motivos zinc finger. O homólogo de TcZFP8 em T. brucei é aparentemente ausente, enquanto um candidato foi identificado em L. major. A clonagem molecular e a expressão heteróloga de TcZFP8 foi realizada para produção de anticorpos e procedimentos como imunocitolocalização, SELEX e EMSA. Análise por Western blotting revelou a presença dessa proteína nas três formas do parasita. Análises usando extratos protéicos nucleares e citoplasmáticos de T.cruzi mostraram que essa proteína está presente na porção nuclear. Esse resultado foi confirmado através de análise de microscopia por imunofluorescência indireta. Experimento de SELEX demonstrou quatro diferentes populações com uma região interna rica em C e/ou G, porém sem seqüências consenso específicas. Análises preliminares de EMSA de uma das quatro populações selecionadas revelaram evidências de que TcZPP8 possa ter afinidade de ligação à fita simples de DNA. __________________________________________________________________________________________ ABSTRACT
Zinc fingers are compact protein domains composed of a α-helix and a β-sheet held together by a zinc ion. Tandem arrays of zinc fingers are commonly used to recognize nucleic acids. Among other activities, they are involved in the processes of replication, transcription, and DNA repair. The nucleocapsid protein of HIV-1 contains a zinc finger motif CX2CX4HX4C that contributes to multiple steps of the viral life cycle, including the proper encapsidation of HIV RNA. In trypanosomatids, only a few of the proteins that contain such fingers were identified. In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX we identified three tandemly linked genes coding for CX2CX4HX4C zinc finger proteins. We also showed that similar genes are present in Trypanosoma brucei and Leishmania major sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Homologous of TcZFP8 in T. brucei is apparently absent, while one candidate in L. major was identified. Molecular cloning of gene TcZFP8 and heterologous expression were performed in Escherichia coli. The purified recombinant protein His6x-TcZFP8 was used to produce antibody in rabbits and GST-TcZFP8 in SELEX and EMSA procedures. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. Analysis using cytoplasm and nuclear cell extracts showed that this protein is present in the nuclear extracts and indirect immunofluorescence microscopy analysis confirmed the nuclear localization of the TcZFP8. SELEX experiment showed four different populations rich in C and/or G nucleotides, but with none consensus sequence. Preliminary EMSA from one population gave evidence that TcZFP8 has affinity to bind to singlestranded DNA.
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Berardi, A. "STRUCTURAL INSIGHTS INTO THE INTERACTION BETWEEN THE TANDEM PHD FINGER DOMAIN P5C5 OF NSD1 AND THE ZINC FINGER MOTIF C2HR OF NIZP1". Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/247139.
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Point Mutations or translocation in NSD1 cause the overgrowth disorder Sotos syndrome and acute myeloid leukemia (AML), respectively (Berdasco M. et al, 2009, Wang G et al, 2003). NSD1 contains several chromatin related domains including a SET domain responsible for histone methyltranferases activity (H3K36 and H4K20), two nuclear receptor-interaction (NID) motifs, five zinc finger domains (PHD1-5), a variant PHD finger (C5HCH), two proline-tryptophan-proline-tryptophan (PWWP1-2) domains (Lucio-Eterovic AK, et al , 2011), suggesting a role in chromatin regulation and gene expression. 20 pathological Sotos mutations have been detected on the PHD tandem domain composed by NSD1-PHD5 and NSD1-C5HCH (NSD1-P5C5). The tandem domain is essential for the pathogenesis of acute myeloid leukaemia (AML) caused by the chimeric protein NUP98/NSD1 that forces the abnormal activation of Hox-A and Meis1 genes (Wang at al 2007). The deletion of this tandem domain is sufficient to abolish NUP98/NSD1 interaction with chromatin, preventing both the transcription activation of HOX genes and the immortalization of myeloid progenitors. The biological role of NSD1-P5C5 is still unclear. It was proposed that this tandem domain is involved in the recognition of both H3K4me3 and H3K9me3 histone marks, (Pasillas M et al. 2011). However, biophysical experiments in our laboratory did not confirm these results challenging the idea that this tandem domain can really work as epigenetic reader. Previous biochemical studies suggested that NSD1-P5C5 can also work as protein-protein interaction motif, being able to bind to the co-repressor Nizp1 by its C2HR zinc fingers motif (Nizp1-C2HR) thus mediating gene repression (Nielsen AL et al, 2004).
The structural determinants of this interaction are still unknown and have been object of this thesis. In order to get more insights into the physiological and pathological role of NSD1-P5C5, we have solved its (i) solution structure by NMR spectroscopy and (ii) characterized its interaction with Nizp1-C2HR.
NSD1-P5C5 folds as unique functional unit adopting a “face to side orientation”. In particular NSD1-PHD5 (or NSD1-P5) presents the canonical PHD finger fold, whereas the NSD1-C5HCH (or NSD1-C5) domain displays an atypical topology characterized by the presence of an additional two stranded β-sheet. In order to investigate the impact of Sotos point mutation on NSD1-P5C5 we expressed and purified seven mutants and analyzed them by NMR. The majority of them destabilize the fold, with the exception of the solvent exposed mutation Arg2152Gln and His2205Arg suggesting a functional role for these residues.
We next solved the solution structure of the zinc finger Nizp1-C2HR, an atypical Cys2His2-type zinc finger in which the fourth zinc chelating residue is substituted by an arginine residue. Its fold consists of a short α-helix and of a short two-stranded β-sheet hold together by one zinc ion. Importantly, we showed that three zinc ligands are sufficient to maintain the protein domain fold and functionality.
NMR titrations of 15N labelled NSD1-P5C5 with Nizp1-C2HR and 15N labelled Nizp1-C2HR with NSD1-P5C5 clearly show that the two proteins directly interact. Analysis of the chemical shift displacements upon complex formation allowed to identify the residues of the two protein domains involved in protein-protein interaction. The interaction surface is located on the interface between NSD1-P5 and NSD1-C5 and on the α-helix of Nizp1-C2HR, respectively.
Based on this information using the software HADDOCK we have computed a data driven docking model of the protein complex. In the model Nizp1-C2HR places its α-helix in the groove at the interface between NSD1-P5 and NSD1-C5, creating both hydrophobic and polar intermolecular contacts.
The thermodynamic parameters that govern complex formation were studied by ITC titrations: the binding reaction is entropy-driven, with a stoichiometry of 1:1 and a Kd of 3,80±0,66 μM. In order to solve the structure of the protein complex we performed crystallographic screenings, and we have found preliminary conditions for obtaining single crystals.
In conclusion, the presented results provide novel information on the interaction between a tandem PHD finger domain and zinc finger motif. The results represent, to the best of our knowledge, the first biophysical characterization between two zinc binding domains. Most importantly, these data give the first molecular details of the interaction between NSD1 and Nizp1 and may provide useful insights into the function of NSD1 and its role in pathological conditions both in Sotos Syndrome and AML. Future work will be dedicated to the full three-dimensional characterization of the complex and to the analysis of Sotos mutations on complex formation.
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John, Rosalind. "Identification of potential gene coding sequences within large cloned DNA arrays : analysis of zinc finger motif". Thesis, Imperial College London, 1991. http://hdl.handle.net/10044/1/46844.
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Purcell, Jamie, i Jamie Purcell. "Investigating the RNA Binding Domains of MBNL1 and the Alternative Splicing Motifs They Recognize". Thesis, University of Oregon, 2012. http://hdl.handle.net/1794/12331.
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Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA binding protein that regulates the alternative splicing of a variety of transcripts. In Myotonic Dystrophy (DM) aberrant cellular localization of MBNL1 results in disease-associated mis-splicing of several MBNL1 target pre-mRNAs. Due to its role in DM pathogenesis, MBNL1 has been a topic of intense study for the last decade, however many open mechanistic questions remain regarding how MBNL1 recognizes RNA substrates to mediate splicing.
The RNA recognition motif for MBNL1, 5'-YGCY-3', was defined herein. This motif was used to identify novel MBNL1 binding sites within regulated transcripts and create synthetic MBNL1-regulated splicing reporters. MBNL1 contains four zinc finger (ZF) RNA binding domains arranged into two pairs of two ZFs. A comprehensive, combinatorial mutagenic study of MBNL1 was conducted to determine the role of each ZF in RNA binding and splicing activity. Functional analysis of the mutant proteins in cellular splicing assays and assessment of RNA binding activity demonstrated that the ZF pairs (i.e. ZF1-2 or ZF3-4) do not have equivalent activity. The ZF1-2 pair is responsible for MBNL1's high affinity RNA binding and splicing activity, whereas the ZF3-4 pair has reduced affinity for RNA and impaired ability to regulate splicing of some transcripts. Hierarchical clustering analysis revealed that two distinct classes of MBNL1-regulated splicing events exist within the small set of splicing events examined. For Class II splicing events the binding and splicing activity for the ZF mutants correlated well. However, for Class I events there was no significant correlation between RNA binding and splicing activity. For pre-mRNAs in the latter class it appears that MBNL1 exerts surprisingly robust splicing activity in the absence of strong RNA binding, suggesting that MBNL1 may be recruited to some pre-mRNA substrates through protein-protein interactions. This study provides the first demonstration that functionally distinct classes of MBNL1-mediated splicing events exist in terms of requirements for different ZFs and the importance of RNA binding.
This dissertation includes previously published and unpublished co-authored material as well as recently co-authored material that has been submitted for publication.
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Kondekar, Sumedha. "Untangling the Helicobacter pylori genome: Deciphering the significance of zinc finger motif(s) in Topoisomerase I catalysis". Thesis, 2018. https://etd.iisc.ac.in/handle/2005/4879.
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Helicobacter pylori, a human pathogen dominating gastric microbial population, displays differential gene expression during various stages of stomach colonization. Topoisomerases play a crucial role in maintaining DNA superhelicity and therefore gene expression. H. pylori has only two topoisomerases: DNA gyrase and Topoisomerase I, as opposed to four in most other prokaryotes. The current study focuses on studying the biochemical and mechanistic details of H. pylori Topoisomerase I (HpTopoI) catalysis. Sequence comparison of HpTopoI with EcTopoI shows the presence of four zinc finger motifs (ZFs) at the carboxyl terminal domain (CTD) unlike three in EcTopoI. To understand the role of ZFs in HpTopoI function, the ZFs were sequentially deleted from the carboxyl terminus. It was observed that the third and fourth ZFs are dispensable for HpTopoI function. DNA relaxation activity was hampered considerably when only one zinc finger (ZF1) was present. Deletion of all ZFs, however, drastically reduced DNA binding and abolished DNA relaxation. These results highlight the importance of ZF1 in catalyzing the relaxation of DNA.
Furthermore, the annotated active site tyrosine residue in HpTopoI when mutated to phenylalanine retained its DNA relaxation activity. Intriguingly, the CTD HpTopoI alone (all four ZFs) could relax the supercoiled DNA although with a specific activity 8.28 fold less than the WT. Gel filtration chromatography analysis suggests that the CTD occurs as dimer in solution and has an ability to form multimers as shown by glutaraldehyde crosslinking. The amino terminal ‘Toprim’ domain houses an acidic triad DxDxE which co-ordinates a Mg2+ cation that is indispensable for the re-ligation activity during the DNA relaxation process. The deletion of Toprim domain or the triple mutation of the acidic triad residues DxDxE to AxAxE reduced the DNA relaxation activity by 14 fold and 9.5 fold, respectively. HpTopoI has a total of 31 tyrosines. An HpTopoI mutant with all the tyrosines mutated to phenylalanine was over-expressed and purified. This mutant is completely inactive which reinstates the fact that it is indeed tyrosine(s) which bring out the nucleophilic attack on the DNA which is essential for DNA relaxation.
A comprehensive analysis of 77 fully sequenced strains of H. pylori revealed the presence of multiple copies of HpTopoI. Out of 77 strains, H. pylori strain XZ274 has emerged as a unique paradigm. This strain was isolated from a gastric cancer patient from Tibet. It has 3 genes annotated as TopoI. This strain lacks a FL copy of HpTopoI. Intriguingly, one of the variant is 317 bp in length encodes for protein with only two zinc fingers.
Topoisomerases have been known to interact with several proteins involved in replication, transcription and recombination. In H. pylori, DprA is a crucial protein involved in natural transformation. This study shows that HpDprA stimulates HpTopoI activity at a lower concentration and interacts physically with HpTopoI as demonstrated by SPR and microscale thermophoresis. This indicates a possible role of HpTopoI during the process of natural transformation.
Taken together, this study reports the biochemical characterization of HpTopoI and sheds light on the unusual role of zinc finger motifs in enzyme catalysis. Analysis of redundant TopoI copies across several H. pylori strains corroborates with the in vitro results that HpTopoI CTD alone can function as a DNA relaxase
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Rivlin, Anatoly A. "Study of mutations in the zinc finger motif of yeast ribosomal protein YL37a /". 1999. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:9951832.
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"Identificação e caracterização de uma proteína com motivos zinc finger de Trypanosoma cruzi". Tese, Biblioteca Digital de teses e Dissertações da UnB, 2006. http://bdtd.bce.unb.br/tedesimplificado/tde_busca/arquivo.php?codArquivo=449.
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Tripathi, Pankaj. "Selective Binding Of Meiosis-Specific Yeast Hop1 Protein, or Its ZnF Motif, To The Holliday Junction Distorts The DNA Structure : Implications For Junction Migration And Resolution". Thesis, 2008. https://etd.iisc.ac.in/handle/2005/894.
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Saccharomyces cerevisiae HOP1, which encodes a component of the synaptonemal complex, plays an important role in both gene conversion and crossing over between homologs, as well as enforces the meiotic recombination checkpoint control over the progression of recombination intermediates. The zinc-finger motif (Znf) 348CX2CX19CX2C374) of Hop1 is crucial for its function in meiosis, since mutation of conserved Cys371 to Ser in this motif results in a temperature-sensitive phenotype, which is defective in sporulation and meiosis. The direct role for Hop1 or its ZnF in the formation of joint molecules and checkpoint control over the progression of meiotic recombination intermediates is unknown. To understand the underlying biochemical mechanism, we constructed a series of recombination intermediates. Hop1 or its ZnF were able to bind different recombination intermediates. Interestingly, the binding affinity of Hop1 and its ZnF was much higher for the Holliday junction as compared to other recombination intermediates. The complexes of Hop1 or its ZnF with the Holliday junction were stable and specific as shown by NaCl titration and competition experiment. Hop1 and its ZnF blocked BLM helicase-induced unwinding of the Holliday junction, indicating that the interaction between Hop1 and its ZnF with the Holliday junction is specific. DNase I footprinting experiment showed that Hop1 or its ZnF bind to the center of the Holliday junction. 2-aminopurine fluorescence and KMnO4 experiments showed that Hop1 or its ZnF can distort the Holliday junction in a 2-fold symmetrical manner. The molecular modeling study showed that Hop1 ZnF folded into unique helix-loop-helix motif and bound to center of the Holliday junction. In summary, this study shows that Hop1 protein or its ZnF interact specifically with the Holliday junction and distort its structure. Taken together, these results implicate that Hop1 protein might coordinate the physical monitoring of meiotic recombination intermediates during the process of branch migration and that Hop1 ZnF acts as a structural determinant of Hop1 protein functions.
Części książek na temat "Zinc finger motifs"
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Hudson, Laurie G., Karen L. Cooper, Susan R. Atlas, Brenee S. King i Ke Jian Liu. "Arsenic Interaction with Zinc Finger Motifs". W Arsenic, 289–314. Hoboken, NJ: John Wiley & Sons, Inc, 2015. http://dx.doi.org/10.1002/9781118876992.ch13.
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Mély, Yves, Etienne Piémont, Monica Sorinas-Jimeno, Hugues de Rocquigny, Nathalie Jullian, Nelly Morellet, Bernard P. Roques i Dominique Gerard. "Structure and tRNAPhe-Binding Properties of the Zinc Finger Motifs of HIV-1 Nucleocapsid Protein". W NATO ASI Series, 369–71. Boston, MA: Springer US, 1994. http://dx.doi.org/10.1007/978-1-4899-1349-4_31.
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de Rocquigny, H., Y. Mely, C. Z. Dong, D. Ficheux, D. Gérard, J. L. Darlix, M. C. Fournié-Zaluski i B. P. Roques. "Interaction of the replication primer tRNA Lys with the HIV-1 nucleocapsid protein NCp7:Structural properties of zinc finger motifs monitored by fluorescence measurements". W Peptides, 777–79. Dordrecht: Springer Netherlands, 1994. http://dx.doi.org/10.1007/978-94-011-0683-2_258.
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Rhodes, D., i A. Klug. "“Zinc Fingers”: A Novel Motif for Nucleic Acid Binding". W Nucleic Acids and Molecular Biology, 149–66. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-83384-7_9.
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Hu, Xiao-Yu, Xiang-Qun Li i Rui Wang. "Studies on the synthesis and DNA-binding activity of zinc finger motif". W Peptides, 28–30. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-010-9069-8_6.
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Kobs-Conrad, Susan F., Ann Marie DiGeorge, Hyosil Lee i Pravin T. P. Kaumaya. "Folding and immunogenicity of a loop-structured peptide using the zinc-finger motif". W Peptides 1992, 561–62. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-011-1470-7_252.
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STRUHL, KEVIN. "HELIX-TURN-HELIX, ZINC-FINGER, AND LEUCINE-ZIPPER MOTIFS FOR EUKARYOTIC TRANSCRIPTIONAL REGULATORY PROTEINS". W Proteins: Form and Function, 259–66. Elsevier, 1990. http://dx.doi.org/10.1016/b978-1-85166-512-9.50032-7.
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Winblad, Stefan, i Anne-Berit Ekström. "Myotonic Dystrophy". W Cognitive and Behavioral Abnormalities of Pediatric Diseases. Oxford University Press, 2010. http://dx.doi.org/10.1093/oso/9780195342680.003.0057.
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The myotonic dystrophies, type 1 (DM1) and 2 (DM2) are progressive, autosomal, dominantly inherited disorders, mainly characterized by muscle weakness and atrophy but also by a variable impact on heart, eye, brain, and the endocrine and the gastrointestinal system (Meola 2000). The worldwide prevalence is approximately 1 in 8,000. They are considered to be most common in Western Europe and Japan, but less prevalent in Southeast Asia, and rare or absent in southern and central Africa (Emery 1991). A prevalence of 18 in 340,000 children has been reported (Darin and Tulinius 2000). The cause of myotonic dystrophies is an unstable inherited repeat DNA expansions. Expansions are elements occurring and repeated throughout the human genome, typically polymorphic in the general population. Repeats can become unstable during DNA replication and, depending on specific repeat motif and location, expanded repeats can become pathogenic. In disease states, the number of repeats exceeds the normal range, leading to various pathogenic mechanisms (Ranum and Cooper 2006). DM1 is associated with an expanded (CTG)n repeat (>50 to several thousands) within the noncoding 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene on chromosome 19q13.3. In DM2, another mutation exists, namely an expanded CCTG tetranucleotide repeat (from 75 to 11,000 repeats) in the first intron of the zinc finger protein 9 (ZNF9) gene on chromosome 3q21 (Day and Ranum 2005). This means that two unrelated genes are associated with similar phenotypes although there are differences, including the age of onset and severity of symptoms (Meola 2000). The first signs of a DM2 disease are typically shown in adulthood, and no study has as yet systematically described cognitive or behavioral abnormalities in a childhood DM2 phenotype. Consequently, the following chapter focuses on a description of DM1. In this disorder, the age of onset is variable, meaning that there are congenital cases, as well as children, adults and patients experiencing the first symptoms very late in life. DM1 is traditionally divided into categories, each presenting with specific clinical features and broadly associated with the age of onset and extent of genetic abnormality.
Streszczenia konferencji na temat "Zinc finger motifs"
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Lihu, Andrei, i Stefan Holban. "The influence of the background model on DNA motif prediction: An assessment for zinc finger transcription factor ZFX". W 2015 IEEE 10th Jubilee International Symposium on Applied Computational Intelligence and Informatics (SACI). IEEE, 2015. http://dx.doi.org/10.1109/saci.2015.7208216.
Pełny tekst źródłaRaporty organizacyjne na temat "Zinc finger motifs"
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Gmeiner, William H. Metal Occupancy of Zinc Finger Motifs as Determinants for Zn2+-Mediated Chemosensitization of Prostate Cancer Cells. Fort Belvoir, VA: Defense Technical Information Center, grudzień 2013. http://dx.doi.org/10.21236/ada596731.
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