RESUMO
Whereas the histone acetyltransferase activity of yeast Gcn5p has been widely studied, its structural interactions with the histones and the role of the carboxy-terminal bromodomain are still unclear. Using a glutathione S-transferase pull down assay we show that Gcn5p binds the amino-terminal tails of histones H3 and H4, but not H2A and H2B. The deletion of bromodomain abolishes this interaction and bromodomain alone is able to interact with the H3 and H4 N termini. The amino acid residues of the H4 N terminus involved in the binding with Gcn5p have been studied by site-directed mutagenesis. The substitution of amino acid residues R19 or R23 of the H4 N terminus with a glutamine (Q) abolishes the interaction with Gcn5p and the bromodomain. These residues differ from those known to be acetylated or to be involved in binding the SIR proteins. This evidence and the known dispensability of the bromodomain for Gcn5p acetyltransferase activity suggest a new structural role for the highly evolutionary conserved bromodomain.
Assuntos
Sequência Conservada , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Arginina , Sítios de Ligação , Proteínas de Ciclo Celular , Evolução Molecular , Glutamina , Histona Acetiltransferases , Modelos Biológicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Transativadores/genética , Fatores de Transcrição , Fatores de Transcrição de p300-CBPRESUMO
Expression of the rolB gene of Agrobacterium rhizogenes TL-DNA is sufficient to trigger root differentiation in transformed plant cells. To investigate the role of RolB in differentiation, a large portion of rolB, comprising about 90% of its C-terminal coding sequence, was cloned into vectors pEX34 and pEA305 in frame with the truncated N termini of the pL-MS2 phage DNA polymerase and, respectively, the ptac-c Its phage lambda repressor gene. Hybrid proteins were expressed from both fusions and the one from pMTBEX1 was utilized to raise antibodies. These antibodies specifically recognize the RolB moiety in both pL-MS2-rolB and ptac-cI-rolB fusions. Unfused, complete RolB protein was obtained by in vitro translation in a rabbit reticulocyte system of a transcript obtained by in vitro transcription of rolB. RolB protein is specifically immunoprecipitated by the antibodies raised against the hybrid protein MS2-RolB.
Assuntos
Anticorpos , Proteínas de Bactérias/genética , Escherichia coli/genética , Genes Bacterianos , Rhizobium/genética , Complexo Antígeno-Anticorpo , Proteínas de Bactérias/análise , Proteínas de Bactérias/imunologia , Western Blotting , Clonagem Molecular/métodos , Expressão Gênica , Peso Molecular , Plasmídeos , Mapeamento por RestriçãoRESUMO
The eukaryotic transposable element Ty1 is present in about 20-30 integrated copies per yeast aploid genome, variably localized in different strains. Here, we report the presence in yeast of circular extrachromosomal molecules homologous to Ty1, 6 kilobases in size (the same as integrated copies) present in about 1 circular copy/250-300 cells. This finding shows another analogy between eukaryotic-transposable elements and the pro-viral integrative form of retroviruses.
Assuntos
Elementos de DNA Transponíveis , DNA Circular/isolamento & purificação , DNA Fúngico/isolamento & purificação , Saccharomyces cerevisiae/genética , Fenômenos Químicos , Química , Saccharomyces cerevisiae/metabolismoRESUMO
Reversible modification of histone tails is a regulatory step in chromatin remodeling. The N-terminal tails of histones are signaling platforms that carry amino acid residues for post-translational modification and contribute to chromosomal higher order structure. These modifications are performed by a number of chromatin modulators such as histone (h) acetyltransferase, h-deacetylase, h-methyltransferase and h-kinase. Large numbers of these enzymes as well as other chromatin-associated proteins share the bromodomain, a signature protein motif. Structural studies reveal not only wide structural conservation of bromodomains but also envision a possible role of this domain in the recognition of specific modified residues in the histone tails. The widespread presence of bromodomains in leukemogenic and cancer genes has provided a fundamental tool for studies of the role of epigenetic and chromatin remodeling in malignant diseases.
Assuntos
Cromatina/metabolismo , Lisina/análogos & derivados , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae , Acetiltransferases/química , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/genética , Histona Acetiltransferases , Humanos , Lisina/metabolismo , Dados de Sequência Molecular , Mutação , Neoplasias/genética , Proteínas de Fusão Oncogênica/fisiologia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Long single-stranded DNA molecules have been observed at electron microscope in DNA preparations from synchronized Chinese hamster cells. The amount of single strandedness in parental DNA increases following a prolonged block of DNA synthesis by hydroxyurea as judged by the results obtained using an improved hydroxyapatite chromatography (Hanania et al., 1975). As far as newly replicated DNA is concerned, an increase of the single strand amount has been observed in DNA preparations from cells actively synthesizing DNA.
Assuntos
Ciclo Celular , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Animais , Células Cultivadas , Cricetinae , Cricetulus , Microscopia EletrônicaRESUMO
Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling. Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates. For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription. P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation. However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero. P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity. Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density. Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription.
Assuntos
DNA Circular/genética , DNA Super-Helicoidal/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Elementos de DNA Transponíveis , Cinética , Plasmídeos , Moldes Genéticos , Transcrição GênicaRESUMO
The alkaline preparation of prokaryotic plasmids (Birnboim and Doly, 1979) has been here adapted to yeast. By simple denaturation and renaturation steps we recovered, from Saccharomyces cerevisiae and Schizosaccharomyces pombe, a population of nucleic acid molecules highly enriched in circular forms. In S. cerevisiae killer strains it is possible to copurify double stranded RNA molecules. The overall recovery was estimated to be 10-30% of the total circular molecules.
Assuntos
DNA Circular/isolamento & purificação , DNA Fúngico/isolamento & purificação , Plasmídeos , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Schizosaccharomyces/genética , Eletroforese em Gel de Ágar/métodos , Concentração de Íons de Hidrogênio , Desnaturação de Ácido Nucleico , Renaturação de Ácido NucleicoRESUMO
The T-regions of the three so far identified types of Ri plasmids-corresponding to the synthesis of three different hairy root opines, agropine, mannopine and cucumopine-have been compared in detail by Southern blot cross hybridizations. Two distinct zones of very strong sequence homology, approximately 4 and 3 kilobases in length respectively, have been identified in all three T-regions. The highly conserved sequences, not present in Ti plasmid T-DNA, may encode essential rhizogenic functions common to all Agrobacterium rhizogenes T-DNAs.
RESUMO
Gcn5p, the nuclear histone acetyltransferase (HAT A), is a component of the multiprotein adaptor complex, ADA. Its role as a transcriptional coactivator is required for full induction of most of the genes regulated by GCN4. In this study we present experimental evidence demonstrating that, during gene activation, the nuclease sensitive region of HIS3 promoter, harbouring the poly (dA:dT) and the GCN4 binding site, is invaded by nucleosomes in a gcn5 disrupted strain. These data demonstrate, for the first time, that Gcn5p affects directly the chromatin organization of a chromosomal gene during its transcriptional activation.
Assuntos
Cromatina/ultraestrutura , Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Hidroliases/genética , Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , DNA Fúngico , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases , Modelos Genéticos , Mutação , Nucleossomos , Proteínas Quinases/genética , Ativação Transcricional , LevedurasRESUMO
Glutamate synthase (GOGAT) and glutamine synthetase play a crucial role in ammonium assimilation and glutamate biosynthesis in the yeast Saccharomyces cerevisiae. The GOGAT enzyme has been purified and the GOGAT structural gene (GLT1) has been cloned, showing that this enzyme is a homotrimeric protein with a monomeric size of 199 kDa. We report the GLT1 nucleotide sequence and the amino acid sequence of its deduced protein product. Our results show that there is a high conservation with the corresponding genes of Escherichia coli, Medicago sativa (alfalfa) and Zea mais (maize). Binding domains for glutamine, cofactors (FMN and NADH) and the cysteine clusters (which comprise the iron-sulfur centres) were tentatively identified on the basis of sequence comparison with GOGAT sequences from E. coli, alfalfa and maize.
Assuntos
Glutamato Sintase/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cisteína/genética , Processamento Eletrônico de Dados , Escherichia coli/genética , Mononucleotídeo de Flavina/genética , Medicago sativa/genética , Dados de Sequência Molecular , NAD/genética , Análise de Sequência , Homologia de Sequência de Aminoácidos , Zea mays/genéticaRESUMO
Truffles form a group of plant-symbiotic Ascomycetes whose hypogeous life cycle is poorly understood. Here we present initial evidence for the influence of light on Tuber borchii mycelial growth and the identification and cloning of a gene, Tbwc-1, homologous to a blue-light photoreceptor of Neurospora crassa. Blue-light irradiation of T. borchii colonies inhibits their apical growth. It also alters apical growth in N. crassa. In Neurospora, the response is controlled by a nuclear photoreceptor, NcWC-1 (White Collar-1), which consists of a sensor domain (LOV) and a transcriptional factor moiety. We isolated a gene (Tbwc-1) whose deduced amino acid sequence shows a high similarity and colinearity of domains with NcWC-1, except for the polyglutamine regions. As previously found in Neurospora, Tbwc-1 mRNA is under light control and its steady state level increases upon irradiation. In silico analysis of the TbWC-1 sensor domain (LOV) supports the hypothesis that TbWC-1 is a photoreceptor, while the absence of the two polyglutamine regions involved in transcriptional activation in Neurospora suggests that this function in Tuber could be lost.
Assuntos
Ascomicetos/crescimento & desenvolvimento , Ascomicetos/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Ascomicetos/citologia , Clonagem Molecular , Sequência Conservada , DNA Fúngico/química , DNA Fúngico/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Luz , Modelos Moleculares , Dados de Sequência Molecular , Morfogênese , Micélio/genética , Micélio/crescimento & desenvolvimento , Neurospora crassa/genética , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/isolamento & purificação , Ácido Poliglutâmico/genética , Estrutura Terciária de Proteína , RNA Fúngico/análise , RNA Mensageiro/análise , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Transcrição GênicaRESUMO
Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression. A collection of increasingly larger 5' deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation. The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5' deletion derivatives. Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Glutamato Sintase/genética , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Glutamato Sintase/química , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.