RESUMO
Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD(+)-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm.
Assuntos
Chlamydomonas reinhardtii/enzimologia , Lactato Desidrogenases/metabolismo , Piruvatos/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/genética , Cloroplastos/enzimologia , Cloroplastos/genética , Fermentação , Lactato Desidrogenases/genética , Modelos Biológicos , Modelos Estruturais , Oxirredução , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismoRESUMO
We have used single-molecule fluorescence microscopy to study the folded state of human telomerase RNA (hTR). Here we show that hTR adopts a new conformation on binding to human telomerase reverse transcriptase (hTERT) and reconstitution of an active ribonucleoprotein complex. Our data are consistent with the formation of an RNA pseudoknot in active human telomerase.
Assuntos
Biocatálise , Conformação de Ácido Nucleico , RNA/química , Telomerase/química , Telomerase/metabolismo , Sequência de Bases , Ativação Enzimática , Humanos , Dados de Sequência Molecular , RNA/síntese química , RNA/metabolismo , Telomerase/síntese química , Telomerase/genéticaRESUMO
It has been proposed that human telomerase RNA (hTR) interacts with dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a dyskerin.hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with dyskerin. We then investigated mutated forms of hTR and dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the dyskerin.hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the dyskerin.hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the dyskerin.hTR interaction may contribute to X-linked DC.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Disceratose Congênita/genética , Mutação/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , RNA/química , RNA/metabolismo , Telomerase/química , Telomerase/metabolismo , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Enzimas de Restrição do DNA/química , Humanos , Microscopia Confocal , Proteínas Nucleares/genética , Mutação Puntual/genética , Ligação Proteica/genética , RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Processamento de Sinais Assistido por Computador , Espectrometria de Fluorescência , Telomerase/genética , TransfecçãoRESUMO
We describe a novel synthetic small molecule which shows an unprecedented stabilization of the human telomeric G-quadruplex with high selectivity relative to double-stranded DNA. We report that this compound can be used in vitro to inhibit telomerase activity and to uncap human POT1 (protection of telomeres 1) from the telomeric G-overhang. We also show that the small molecule G-quadruplex binder induces a partial alteration of shelterin through POT1 uncapping from telomeres in human HT1080 cancer cells and the presence of gammaH2AX foci colocalized at telomeres.
Assuntos
Dano ao DNA/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo , Telômero/química , Telômero/metabolismo , Linhagem Celular Tumoral , Humanos , Estrutura Molecular , Complexo Shelterina , Telômero/genética , Proteínas de Ligação a Telômeros/antagonistas & inibidoresRESUMO
The novel adduct 1,1,3,3-tetramethylguanidine-gallane, (Me2N)2CN(H).GaH3, has been prepared by the reaction of [(Me2N)2CNH2]+Cl- with LiGaH4 in Et2O solution. Its spectroscopic properties indicate a monomeric species with an unusually strong coordinate link between the imido function and GaH3, an inference confirmed by the crystal structure at 150 K which also reveals significant secondary interactions through non-classical N-H...H-Ga bridges. Despite the intrinsic strength of the Ga-N bond, however, vaporisation at ca. 310 K occurs with partial dissociation, and decomposition via more than one pathway proceeds at temperatures >330-350 K to give a variety of products, including the free base, Me2NH, H2, and a novel gallium-nitrogen compound composed of a Ga4N4 cubane-like core bridged on three edges by -N{C(NMe2)2}GaH2- units.