Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Plant Cell Physiol ; 60(8): 1633-1645, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292642

RESUMO

Plants respond to a rise in ambient temperature by increasing the growth of petioles and hypocotyls. In this work, we show that Arabidopsis thaliana class I TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP) transcription factors TCP14 and TCP15 are required for optimal petiole and hypocotyl elongation under high ambient temperature. These TCPs influence the levels of the DELLA protein RGA and the expression of growth-related genes, which are induced in response to an increase in temperature. However, the class I TCPs are not required for the induction of the auxin biosynthesis gene YUCCA8 or for auxin-dependent gene expression responses. TCP15 directly targets the gibberellin biosynthesis gene GA20ox1 and the growth regulatory genes HBI1 and PRE6. Several of the genes regulated by TCP15 are also targets of the growth regulator PIF4 and show an enrichment of PIF4- and TCP-binding motifs in their promoters. PIF4 binding to GA20ox1 and HBI1 is enhanced in the presence of the TCPs, indicating that TCP14 and TCP15 directly participate in the induction of genes involved in gibberellin biosynthesis and cell expansion by high temperature functionally interacting with PIF4. In addition, overexpression of HBI1 rescues the growth defects of tcp14 tcp15 double mutants, suggesting that this gene is a major outcome of regulation by both class I TCPs during thermomorphogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Giberelinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Temperatura Ambiente , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Plant Physiol ; 170(1): 74-85, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26574599

RESUMO

TCP proteins integrate a family of transcription factors involved in the regulation of developmental processes and hormone responses. It has been shown that most members of class I, one of the two classes in which the TCP family is divided, contain a conserved Cys that leads to inhibition of DNA binding when oxidized. In this work, we describe that the class-I TCP protein TCP15 inhibits anthocyanin accumulation during exposure of plants to high light intensity by modulating the expression of transcription factors involved in the induction of anthocyanin biosynthesis genes, as suggested by the study of plants that express TCP15 from the 35SCaMV promoter and mutants in TCP15 and the related gene TCP14. In addition, the effect of TCP15 on anthocyanin accumulation is lost after prolonged incubation under high light intensity conditions. We provide evidence that this is due to inactivation of TCP15 by oxidation of Cys-20 of the TCP domain. Thus, redox modulation of TCP15 activity in vivo by high light intensity may serve to adjust anthocyanin accumulation to the duration of exposure to high irradiation conditions.


Assuntos
Antocianinas/biossíntese , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fatores de Transcrição/metabolismo , Antocianinas/metabolismo , Proteínas de Arabidopsis/genética , Cisteína/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Mutação , Oxirredução , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Fatores de Transcrição/genética
3.
Plant Physiol ; 162(3): 1434-47, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23686421

RESUMO

TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factors participate in plant developmental processes associated with cell proliferation and growth. Most members of class I, one of the two classes that compose the family, have a conserved cysteine at position 20 (Cys-20) of the TCP DNA-binding and dimerization domain. We show that Arabidopsis (Arabidopsis thaliana) class I proteins with Cys-20 are sensitive to redox conditions, since their DNA-binding activity is inhibited after incubation with the oxidants diamide, oxidized glutathione, or hydrogen peroxide or with nitric oxide-producing agents. Inhibition can be reversed by treatment with the reductants dithiothreitol or reduced glutathione or by incubation with the thioredoxin/thioredoxin reductase system. Mutation of Cys-20 in the class I protein TCP15 abolished its redox sensitivity. Under oxidizing conditions, covalently linked dimers were formed, suggesting that inactivation is associated with the formation of intermolecular disulfide bonds. Inhibition of class I TCP protein activity was also observed in vivo, in yeast (Saccharomyces cerevisiae) cells expressing TCP proteins and in plants after treatment with redox agents. This inhibition was correlated with modifications in the expression of the downstream CUC1 gene in plants. Modeling studies indicated that Cys-20 is located at the dimer interface near the DNA-binding surface. This places this residue in the correct orientation for intermolecular disulfide bond formation and explains the sensitivity of DNA binding to the oxidation of Cys-20. The redox properties of Cys-20 and the observed effects of cellular redox agents both in vitro and in vivo suggest that class I TCP protein action is under redox control in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cisteína/genética , Cisteína/metabolismo , DNA de Plantas/metabolismo , Diamida/metabolismo , Dissulfetos/química , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Modelos Moleculares , Mutação , Oxirredução , Estresse Oxidativo , Multimerização Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
4.
Biomol Concepts ; 4(2): 111-27, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25436570

RESUMO

After its initial definition in 1999, the TCP family of transcription factors has become the focus of a multiplicity of studies related with plant development at the cellular, organ, and tissue levels. Evidence has accumulated indicating that TCP transcription factors are the main regulators of plant form and architecture and constitute a tool through which evolution shapes plant diversity. The TCP transcription factors act in a multiplicity of pathways related with cell proliferation and hormone responses. In recent years, the molecular pathways of TCP protein action and biochemical studies on their mode of interaction with DNA have begun to shed light on their mechanism of action. However, the available information is fragmented and a unifying view of TCP protein action is lacking, as well as detailed structural studies of the TCP-DNA complex. Also important, the possible role of TCP proteins as integrators of plant developmental responses to the environment has deserved little attention. In this review, we summarize the current knowledge about the structure and functions of TCP transcription factors and analyze future perspectives for the study of the role of these proteins and their use to modify plant development.


Assuntos
Desenvolvimento Vegetal/fisiologia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Fatores de Transcrição/metabolismo , Relógios Circadianos , Proteínas de Plantas/genética , Domínios e Motivos de Interação entre Proteínas , Elementos de Resposta , Fatores de Transcrição/genética
5.
Plant Physiol ; 161(2): 760-72, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23221774

RESUMO

BEL1- and KNOTTED1-type proteins are transcription factors from the three-amino-loop-extension superclass that interact in a tandem complex to regulate the expression of target genes. In potato (Solanum tuberosum), StBEL5 and its Knox protein partner regulate tuberization by targeting genes that control growth. RNA movement assays demonstrated that StBEL5 transcripts move through the phloem to stolon tips, the site of tuber induction. StBEL5 messenger RNA originates in the leaf, and its movement to stolons is induced by a short-day photoperiod. Here, we report the movement of StBEL5 RNA to roots correlated with increased growth, changes in morphology, and accumulation of GA2-oxidase1, YUCCA1a, and ISOPENTENYL TRANSFERASE transcripts. Transcription of StBEL5 in leaves is induced by light but insensitive to photoperiod, whereas in stolon tips growing in the dark, promoter activity is enhanced by short days. The heterodimer of StBEL5 and POTH1, a KNOTTED1-type transcription factor, binds to a tandem TTGAC-TTGAC motif that is essential for regulating transcription. The discovery of an inverted tandem motif in the StBEL5 promoter with TTGAC motifs on opposite strands may explain the induction of StBEL5 promoter activity in stolon tips under short days. Using transgenic potato lines, deletion of one of the TTGAC motifs from the StBEL5 promoter results in the reduction of GUS activity in new tubers and roots. Gel-shift assays demonstrate BEL5/POTH1 binding specificity to the motifs present in the StBEL5 promoter and a double tandem motif present in the StGA2-oxidase1 promoter. These results suggest that, in addition to tuberization, the movement of StBEL5 messenger RNA regulates other aspects of vegetative development.


Assuntos
Tubérculos/metabolismo , Transporte de RNA , RNA de Plantas/metabolismo , Solanum tuberosum/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Luz , Motivos de Nucleotídeos/genética , Floema/genética , Floema/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Tubérculos/genética , Tubérculos/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
J Exp Bot ; 63(2): 809-23, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22016421

RESUMO

The function of the class I TCP transcription factor TCP15 from Arabidopsis thaliana has been studied through the analysis of plants that express a fusion of this protein to the EAR repressor domain. Constitutive expression of TCP15-EAR produces growth arrest at the seedling stage, before leaf emergence. Expression of the repressor fusion from the AtTCP15 promoter produces small plants with leaves whose margins progressively curve upwards, starting from the basal part of the lamina. Leaves contain smaller and less differentiated cells, both on the adaxial and abaxial sides. The abaxial domain is relatively enlarged, with disorganized cells separated by empty spaces. TCP15-EAR also affects the growth of leaf petioles, flower pedicels, and anther filaments. Flowers show reduced elongation of the three outer whorls and altered gynoecia with irregular carpel surfaces and enlarged repla. Ectopic stigma-like structures develop from medial and basal parts of the replum. TCP15-EAR produces an increase in expression of the boundary-specific genes LOB, CUC1, and CUC2. Changes in CUC1 and CUC2 expression can be explained by the existence of lower levels of miR164 in leaves and the repression of IAA3/SHY2 and the SAUR-like gene At1g29460 in leaves and flowers. TCP15 binds to the promoter regions of IAA3/SHY2 and At1g29460, suggesting that these genes may be direct targets of the transcription factor. The results indicate that TCP15 regulates the expression of boundary-specific genes through a pathway that affects auxin homeostasis and partially overlaps with the one modulated by class II CIN-like TCP proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Ácidos Indolacéticos/metabolismo , Elementos Isolantes/genética , Fatores de Transcrição/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Cotilédone/anatomia & histologia , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Flores/anatomia & histologia , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Homeostase , MicroRNAs/genética , Mutação , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Brotos de Planta/anatomia & histologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Fatores de Transcrição/genética
7.
J Biol Chem ; 287(1): 347-56, 2012 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-22074922

RESUMO

TCP proteins constitute a family of plant transcription factors with more than 20 members in angiosperms. They can be divided in two classes based on sequence homology and the presence of an insertion within the basic region of the TCP DNA binding and dimerization domain. Here, we describe binding site selection studies with the class I protein TCP16, showing that its DNA binding preferences are similar to those of class II proteins. Through sequence comparison and the analysis of mutants and chimeras of TCP16, TCP20 (class I), and TCP4 (class II), we established that the identity of residue 11 of the class I TCP domain or the equivalent residue 15 of the class II domain, whether it is Gly or Asp, determines a preference for a class I or a class II sequence, respectively. Footprinting analysis indicated that specific DNA contacts related to these preferences are established with one of the strands of DNA. The dimerization motif also influences the selectivity of the proteins toward class I and class II sequences and determines a requirement of an extended basic region in proteins with Asp-15. We postulate that differences in orientation of base-contacting residues brought about by the presence of either Gly or Asp are responsible for the binding site preferences of TCP proteins. Expression of repressor forms of TCP16 with Asp-11 or Gly-11 differently affects leaf development. TCP16-like proteins with Asp-11 in the TCP domain arose in rosids and may be related to developmental characteristics of this lineage of eudicots.


Assuntos
Proteínas de Arabidopsis/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis , Proteínas de Arabidopsis/química , Sequência de Bases , Sítios de Ligação , DNA/genética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Fatores de Transcrição/química
8.
Methods Mol Biol ; 754: 259-75, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21720958

RESUMO

In the following chapter we describe methods and protocols to analyze the interaction of proteins with DNA using footprinting and related techniques based on the modification of DNA with either hydroxyl radicals or methylating agents. Footprinting, based on the protection from chemical modification of DNA through the specific binding of a protein, gives information about the nucleotides that are in close contact with the protein upon binding. The derived missing nucleoside and interference techniques identify nucleotides that are energetically important for protein binding. These methods are highly valuable to study in detail the interaction of a transcription factor with nucleotides on both strands of its target DNA sequence.


Assuntos
Pegada de DNA/métodos , DNA de Plantas/análise , Fatores de Transcrição/análise , Sequência de Bases , Sítios de Ligação/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Radical Hidroxila , Metilação , Modelos Moleculares , Análise de Sequência de DNA/métodos , Coloração e Rotulagem/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Plant Physiol ; 156(4): 1894-904, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21685178

RESUMO

The KNOXI transcription factor SHOOT MERISTEMLESS (STM) is required to establish and maintain the Arabidopsis (Arabidopsis thaliana) apical meristem, yet little is known about its direct targets. Using different approaches we demonstrate that the induction of STM causes a significant up-regulation of the organ boundary gene CUP SHAPED COTYLEDON1 (CUC1), which is specific and independent of other meristem regulators. We further show that the regulation of CUC1 by STM is direct and identify putative binding sites in its promoter. Continuous expression of STM in Arabidopsis leaf primordia also causes the activation of CUC2-3, as well as microRNA MIR164a, which provides a negative feedback loop by posttranscriptionally regulating CUC1 and CUC2. The results bring new insights into the mechanistic links between KNOXI and CUC transcription factors and contribute to the understanding of the regulatory network controlled by STM.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Homeodomínio/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Retroalimentação Fisiológica , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica
10.
Biochem J ; 435(1): 143-55, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21241251

RESUMO

The TCP domain is a DNA-binding domain present in plant transcription factors that modulate different processes. In the present study, we show that Arabidopsis class I TCP proteins are able to interact with a dyad-symmetric sequence composed of two GTGGG half-sites. TCP20 establishes symmetric interactions with the 5' half of each strand, whereas TCP11 interacts mainly with the 3' half. SELEX (systematic evolution of ligands by exponential enrichment) experiments with TCP15 and TCP20 indicated that these proteins have similar, although not identical, DNA-binding preferences and are able to interact with non-palindromic binding sites of the type GTGGGNCCNN. TCP11 shows a different DNA-binding specificity, with a preference for the sequence GTGGGCCNNN. The distinct DNA-binding properties of TCP11 are due to the presence of a threonine residue at position 15 of the TCP domain, a position that is occupied by an arginine residue in most TCP proteins. TCP11 also forms heterodimers with TCP15 that have increased DNA-binding efficiency. The expression in plants of a repressor form of TCP11 demonstrated that this protein is a developmental regulator that influences the growth of leaves, stems and petioles, and pollen development. The results suggest that changes in DNA-binding preferences may be one of the mechanisms through which class I TCP proteins achieve functional specificity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Domínios e Motivos de Interação entre Proteínas , Treonina/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Sequência Consenso , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Glucuronidase/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Pegadas de Proteínas , Multimerização Proteica , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
11.
Biochimie ; 91(8): 974-81, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19442701

RESUMO

We have analyzed the DNA-binding properties of the complex formed by the Arabidopsis TALE homeodomain (HD) proteins STM and BLH3 in comparison with those of the individual proteins. In vitro DNA-binding assays indicated that complex formation increases binding affinity for sequences carrying either a single target site or two such sites arranged in tandem. Complex formation is not correlated with the establishment of new detectable contacts as deduced from missing-nucleoside experiments. Increased binding was also observed when using BLH3 with a mutation that renders the HD unable to bind DNA, suggesting that only the STM functional HD is necessary for tight binding by the complex. Yeast one-hybrid assays using single or double target sites showed that the effect of complex formation is more dramatic for the double target site and that under these conditions competition for binding by the individual proteins is reduced. The results indicate that even if complex formation produces an increase in binding to DNA sequences containing either one or two target sites, the relative increase in binding produced after complex formation is dependent on the type of target sequence that is considered. This differential effect of complex formation on binding may have implications in the regulatory properties of these transcription factors within the cell.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Sequência de Bases , Sítios de Ligação , DNA/genética , Proteínas de Homeodomínio/química , Nucleosídeos/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Fatores de Transcrição/química
12.
Plant Mol Biol ; 69(6): 729-43, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19125337

RESUMO

Arabidopsis COX5b-1 encodes an isoform of the zinc binding subunit 5b of mitochondrial cytochrome c oxidase. A promoter region required for expression and induction by sucrose of this gene was analyzed using plants stably transformed with mutagenized promoter fragments fused to the gus reporter gene. Promoter dependent expression is absolutely dependent on a G-box present at -228 from the translation start site. This element interacts in vitro and in vivo with transcription factors from the bZip family, preferentially with the abscisic acid-responsive element binding factor AREB2/ABF4. A region located upstream of the G-box (-333/-259) contains elements with the core sequence ATCATT and distalB-like sequences (CCACTTG) that are required for expression in vegetative tissues. These sequences bind different sets of proteins present in plant nuclear extracts and participate in induction by sucrose (ATCATT) and abscisic acid (distalB) of the COX5b-1 promoter. We propose that the COX5b-1 promoter has acquired novel regulatory mechanisms during evolution after gene duplication. These novel mechanisms have allowed the diversification of expression patterns, but also the conservation of some responses that, as induction by sucrose, are shared by COX5b-1 and other genes encoding components of the mitochondrial respiratory chain. Conservation of these responses may be a pre-requisite for the successful incorporation of new regulatory elements in this class of genes.


Assuntos
Proteínas de Arabidopsis/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Regiões Promotoras Genéticas/genética , Sacarose/farmacologia , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Sítios de Ligação/genética , Núcleo Celular/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucuronidase/genética , Glucuronidase/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-Híbrido , Zinco/metabolismo
13.
J Exp Bot ; 60(3): 829-45, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19098132

RESUMO

Sequences required for the expression of Cytc-2 (At4g10040), one of two cytochrome c genes from Arabidopsis thaliana, were characterized using plants transformed with deleted and mutagenized promoter fragments fused to gus. These studies indicated that a region containing a G-box and an ACGT motif is essential for expression. Mutation of the ACGT motif causes a complete loss of expression, while mutation of the G-box causes decreased expression in aerial parts and abolishes expression in roots and induction by environmental factors. Upstream located site II elements are required for maximal expression, mainly in reproductive tissues, and maximal induction by different factors. One-hybrid screenings allowed the identification of transcription factors from the bZIP and bHLH families that interact mainly with the G-box. Four of these factors were able to bind to the Cytc-2 promoter in vitro and in transactivation assays in Arabidopsis. Analysis of available microarray data indicated that the bZIP transcription factors share expression characteristics with the Cytc-2 gene, suggesting that they act as mediators of its response to tissue-specific, environmental, and metabolic conditions. Site II elements interact with a TCP family protein and may co-ordinate the expression of the Cytc-2 gene with that of other respiratory chain components. A model is proposed for the evolution of the Cytc-2 gene through the incorporation of a segment containing a G-box and an ACGT motif into an ancestral gene that contained site II elements. This may have reduced the importance of site II elements for basal expression and conferred new responses to environmental factors.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Citocromos c/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , DNA de Plantas/metabolismo , Deleção de Genes , Genes Reporter , Glucuronidase/metabolismo , Dados de Sequência Molecular , Mutagênese , Mutação/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/genética , Fatores de Transcrição/metabolismo
14.
Biochemistry ; 46(25): 7416-25, 2007 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-17530779

RESUMO

HAT3.1 is a member of the PHD-finger homeodomain protein family. The HAT3.1 homeodomain is highly divergent in sequence even at positions that are almost invariable among homeodomains. In this work, we have applied the random oligonucleotide selection technique to investigate if the HAT3.1 homeodomain is able to recognize specific DNA sequences. Analysis of the selected molecules followed by hydroxyl radical footprinting experiments and yeast one-hybrid assays indicated that HAT3.1 shows a preference for the sequence T(A/G)(A/C)ACCA, different from those bound by other homeodomains. Binding was dependent on homeodomain residues located at positions 47, 50, 51, and 54, the same positions that usually participate in DNA binding in most homeodomains. The study of the interaction of mutants at these positions with DNA carrying nucleotide changes at specific sites suggested that H51 and K50 most likely interact with nucleotides 2 to 4 and 5 to 6, respectively, while W54 would establish contacts with position 4. The presence of H51 and W54 represents an innovation among homeodomain structures. The fact that the HAT3.1 homeodomain is able to interact with specific DNA sequences is evidence of the inherent plasticity of the homeodomain as a DNA binding unit.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , DNA de Plantas/metabolismo , Histidina/química , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência de Bases , DNA de Plantas/química , Proteínas de Homeodomínio/genética , Modelos Químicos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
15.
Biol Chem ; 387(1): 31-40, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16497162

RESUMO

We have studied the interaction of the BELL-like Arabidopsis homeodomain protein ATH1 with DNA. Analysis of oligonucleotides selected by the ATH1 homeodomain from a random mixture suggests that ATH1 preferentially binds the sequence TGACAGGT. Single nucleotide replacements at positions 2 or 3 of this sequence abolish binding, while changes at position 4 are more tolerated. Changes outside this core differentially affect binding, depending on the position. Hydroxyl radical footprinting and missing nucleoside experiments showed that ATH1 interacts with a 7-bp region of the strand carrying the GAC core. On the other strand, protection was observed over a 7-bp region, comprising one additional nucleotide complementary to T in position 1. A comparative analysis of the binding preferences of the homeodomains of ATH1 and STM (a KNOX homeodomain protein) indicated that they bind similar sequences, but with differences in affinity and specificity. The decreased affinity displayed by the ATH1 homeodomain correlates with the presence of valine (instead of lysine as in STM) at position 54. This difference also explains the decreased and increased selectivities, respectively, at positions 4 and 5. Our results point to an essential role of residue 54 in determining the different binding properties of BELL and KNOX homeodomains.


Assuntos
Proteínas de Arabidopsis/metabolismo , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência de Bases , Sítios de Ligação/fisiologia , Clonagem Molecular , Pegada de DNA , DNA de Plantas/química , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Radical Hidroxila/química , Dados de Sequência Molecular , Mutação , Ligação Proteica/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
16.
FEBS J ; 272(1): 190-202, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15634343

RESUMO

The interaction of the homeodomain of the sunflower KNOX protein HAKN1 with DNA was studied by site-directed mutagenesis, hydroxyl radical footprinting and missing nucleoside experiments. Binding of HAKN1 to different oligonucleotides indicated that HAKN1 prefers the sequence TGACA (TGTCA), with changes within the GAC core more profoundly affecting the interaction. Footprinting and missing nucleoside experiments using hydroxyl radical cleavage of DNA showed that HAKN1 interacts with a 6-bp region of the strand carrying the GAC core, covering the core and nucleotides towards the 3' end. On the other strand, protection was observed along an 8-bp region, comprising two additional nucleotides complementary to those preceding the core. Changes in the residue present at position 50 produced proteins with different specificities. An I50S mutant showed a preference for TGACT, while the presence of lysine shifted the preference to TGACC, suggesting that residue 50 interacts with nucleotide(s) 3' to GAC. Mutation of Lys54-->Val produced a protein with reduced affinity and relaxed specificity, able to recognize the sequence TGAAA, while the conservative change of Arg55-->Lys completely abolished binding to DNA. Based on these results, we propose a model for the interaction of HAKN1 with DNA in which helix III of the homeodomain accommodates along the major groove with Arg55, Asn51, Lys54 and Ile50, establishing specific contacts with bases of the GACA sequence or their complements. This model can be extended to other KNOX proteins given the conservation of these amino acids in all members of the family.


Assuntos
Pegada de DNA , DNA de Plantas/metabolismo , Proteínas de Homeodomínio/metabolismo , DNA de Plantas/genética , Helianthus , Mutagênese Sítio-Dirigida , Ligação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA