RESUMEN
DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.
Asunto(s)
Arabidopsis , Cloroplastos , Tilacoides , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Tilacoides/metabolismo , Tilacoides/genética , Tilacoides/ultraestructura , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transcripción Genética , ADN de Cloroplastos/genética , ADN de Cloroplastos/metabolismoRESUMEN
DNA methylation is a conserved epigenetic mark in plants and mammals. In Arabidopsis, DNA methylation can be triggered by small interfering RNAs (siRNAs) through an RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification of an RdDM effector, KTF1. Loss-of-function mutations in KTF1 reduce DNA methylation and release the silencing of RdDM target loci without abolishing the siRNA triggers. KTF1 has similarity to the transcription elongation factor SPT5 and contains a C-terminal extension rich in GW/WG repeats. KTF1 colocalizes with ARGONAUTE 4 (AGO4) in punctate nuclear foci and binds AGO4 and RNA transcripts. Our results suggest KTF1 as an adaptor protein that binds scaffold transcripts generated by Pol V and recruits AGO4 and AGO4-bound siRNAs to form an RdDM effector complex. The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA-silencing effector complexes.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Metilación de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Argonautas , Sitios de Unión , ADN de Plantas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. 24-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of â¼ 20-60 nt in length, often having the same 5' ends but differing in 3' ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on distributive 3'-5' exonucleases. Our findings suggest an alternative route for siRNA biogenesis. Precursor transcripts are bound by AGO4 and subsequently subjected to 3'-5' exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilación de ADN , ARN Interferente Pequeño/biosíntesis , Proteínas de Arabidopsis/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Genoma de Planta , Datos de Secuencia Molecular , Mutación/genética , ARN de Planta/genética , ARN Polimerasa Dependiente del ARN/genética , Plantones/genéticaRESUMEN
Plastids contain their own genomes, which are transcribed by two types of RNA polymerases. One of those enzymes is a bacterial-type, multi-subunit polymerase encoded by the plastid genome. The plastid-encoded RNA polymerase (PEP) is required for efficient expression of genes encoding proteins involved in photosynthesis. Despite the importance of PEP, its DNA binding locations have not been studied on the genome-wide scale at high resolution. We established a highly specific approach to detect the genome-wide pattern of PEP binding to chloroplast DNA using plastid chromatin immunoprecipitation-sequencing (ptChIP-seq). We found that in mature Arabidopsis thaliana chloroplasts, PEP has a complex DNA binding pattern with preferential association at genes encoding rRNA, tRNA, and a subset of photosynthetic proteins. Sigma factors SIG2 and SIG6 strongly impact PEP binding to a subset of tRNA genes and have more moderate effects on PEP binding throughout the rest of the genome. PEP binding is commonly enriched on gene promoters, around transcription start sites. Finally, the levels of PEP binding to DNA are correlated with levels of RNA accumulation, which demonstrates the impact of PEP on chloroplast gene expression. Presented data are available through a publicly available Plastid Genome Visualization Tool (Plavisto) at https://plavisto.mcdb.lsa.umich.edu/.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , ADN de Cloroplastos/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes del Cloroplasto , Plastidios/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Factor sigma/genética , Transcripción GenéticaRESUMEN
In this work, a series of novel boronium-bis(trifluoromethylsulfonyl)imide [TFSI-] ionic liquids (IL) are introduced and investigated. The boronium cations were designed with specific structural motifs that delivered improved electrochemical and physical properties, as evaluated through cyclic voltammetry, broadband dielectric spectroscopy, densitometry, thermogravimetric analysis, and differential scanning calorimetry. Boronium cations, which were appended with N-alkylpyrrolidinium substituents, exhibited superior physicochemical properties, including high conductivity, low viscosity, and electrochemical windows surpassing 6 V. Remarkably, the boronium ionic liquid functionalized with both an ethyl-substituted pyrrolidinium and trimethylamine, [(1-e-pyrr)N111BH2][TFSI], exhibited a 6.3 V window, surpassing previously published boronium-, pyrrolidinium-, and imidazolium-based IL electrolytes. Favorable physical properties and straightforward tunability make boronium ionic liquids promising candidates to replace conventional organic electrolytes for electrochemical applications requiring high voltages.
RESUMEN
Nuclear transcription is not restricted to genes but occurs throughout the intergenic and noncoding space of eukaryotic genomes. The functional significance of this widespread noncoding transcription is mostly unknown. We show that Arabidopsis RNA polymerase IVb/Pol V, a multisubunit nuclear enzyme required for siRNA-mediated gene silencing of transposons and other repeats, transcribes intergenic and noncoding sequences, thereby facilitating heterochromatin formation and silencing of overlapping and adjacent genes. Pol IVb/Pol V transcription requires the chromatin-remodeling protein DRD1 but is independent of siRNA biogenesis. However, Pol IVb/Pol V transcription and siRNA production are both required to silence transposons, suggesting that Pol IVb/Pol V generates RNAs or chromatin structures that serve as scaffolds for siRNA-mediated heterochromatin-forming complexes. Pol IVb/Pol V function provides a solution to a paradox of epigenetic control: the need for transcription in order to transcriptionally silence the same region.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Regulación de la Expresión Génica de las Plantas , Regulación de la Expresión Génica , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Cromatina/química , Cromatina/metabolismo , ADN Intergénico , ARN Polimerasas Dirigidas por ADN/metabolismo , Silenciador del Gen , Heterocromatina/química , Datos de Secuencia Molecular , ARN Interferente Pequeño/metabolismo , ARN no Traducido , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Correction for 'Tuning the melting point of selected ionic liquids through adjustment of the cation's dipole moment' by Brooks D. Rabideau et al., Phys. Chem. Chem. Phys., 2020, 22, 12301-12311, https://doi.org/10.1039/D0CP01214A.
RESUMEN
Non-coding transcription is an important determinant of heterochromatin formation. In Arabidopsis thaliana a specialized RNA polymerase V (Pol V) transcribes pervasively and produces long non-coding RNAs. These transcripts work with small interfering RNA to facilitate locus-specific establishment of RNA-directed DNA methylation (RdDM). Subsequent maintenance of RdDM is associated with elevated levels of Pol V transcription. However, the impact of DNA methylation on Pol V transcription remained unresolved. We found that DNA methylation strongly enhances Pol V transcription. The level of Pol V transcription is reduced in mutants defective in RdDM components working downstream of Pol V, indicating that RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription. Pol V transcription is affected only on loci that lose DNA methylation in all sequence contexts in a particular mutant, including mutants lacking maintenance DNA methyltransferases, which suggests that RdDM works in a complex crosstalk with other silencing pathways.
Asunto(s)
Arabidopsis/genética , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Elementos Transponibles de ADN , ADN-Citosina Metilasas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Retroalimentación Fisiológica , Metiltransferasas/metabolismo , ARN Largo no Codificante/biosíntesis , Transcripción GenéticaRESUMEN
Eukaryotic genomes are pervasively transcribed, yet most transcribed sequences lack conservation or known biological functions. In Arabidopsis thaliana, RNA polymerase V (Pol V) produces noncoding transcripts, which base pair with small interfering RNA (siRNA) and allow specific establishment of RNA-directed DNA methylation (RdDM) on transposable elements. Here, we show that Pol V transcribes much more broadly than previously expected, including subsets of both heterochromatic and euchromatic regions. At already established RdDM targets, Pol V and siRNA work together to maintain silencing. In contrast, some euchromatic sequences do not give rise to siRNA but are covered by low levels of Pol V transcription, which is needed to establish RdDM de novo if a transposon is reactivated. We propose a model where Pol V surveils the genome to make it competent to silence newly activated or integrated transposons. This indicates that pervasive transcription of nonconserved sequences may serve an essential role in maintenance of genome integrity.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Genoma , ARN no Traducido , Transcripción Genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Elementos Transponibles de ADN , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Especificidad por SustratoRESUMEN
RNA-mediated transcriptional silencing prevents deleterious effects of transposon activity and controls the expression of protein-coding genes. It involves long noncoding RNAs (lncRNAs). In Arabidopsis thaliana, some of those lncRNAs are produced by a specialized RNA Polymerase V (Pol V). The mechanism by which lncRNAs affect chromatin structure and mRNA production remains mostly unknown. Here we identify the SWI/SNF ATP-dependent nucleosome-remodeling complex as a component of the RNA-mediated transcriptional silencing pathway. We found that SWI3B, an essential subunit of the SWI/SNF complex, physically interacts with a lncRNA-binding protein, IDN2. SWI/SNF subunits contribute to lncRNA-mediated transcriptional silencing. Moreover, Pol V mediates stabilization of nucleosomes on silenced regions. We propose that Pol V-produced lncRNAs mediate transcriptional silencing by guiding the SWI/SNF complex and establishing positioned nucleosomes on specific genomic loci. We further propose that guiding ATP-dependent chromatin-remodeling complexes may be a more general function of lncRNAs.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Interferencia de ARN , ARN Largo no Codificante/genética , ARN de Planta/genética , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Ensamble y Desensamble de Cromatina , Metilación de ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/metabolismo , ARN Largo no Codificante/metabolismo , ARN de Planta/metabolismo , Proteínas de Unión al ARN/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
RNase H1 is an endonuclease specific toward the RNA strand of RNA:DNA hybrids. Members of this protein family are present in most living organisms and are essential for removing RNA that base pairs with DNA. It prevents detrimental effects of RNA:DNA hybrids and is involved in several biological processes. Arabidopsis thaliana has been previously shown to contain three genes encoding RNase H1 proteins that localize to three distinct cellular compartments. We show that these genes originate from two gene duplication events. One occurred in the common ancestor of dicots and produced nuclear and organellar RNase H1 paralogs. Second duplication occurred in the common ancestor of Brassicaceae and produced mitochondrial- and plastid-localized proteins. These proteins have the canonical RNase H1 activity, which requires at least four ribonucleotides for endonucleolytic digestion. Analysis of mutants in the RNase H1 genes revealed that the nuclear RNH1A and mitochondrial RNH1B are dispensable for development under normal growth conditions. However, the presence of at least one organellar RNase H1 (RNH1B or RNH1C) is required for embryonic development. The plastid-localized RNH1C affects plastid DNA copy number and sensitivity to replicative stress. Our results present the evolutionary history of RNH1 proteins in A. thaliana, demonstrate their canonical RNase H1 activity and indicate their role in early embryonic development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/enzimología , Ribonucleasa H/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassicaceae/enzimología , Brassicaceae/genética , Cloroplastos/enzimología , Cloroplastos/metabolismo , Evolución Molecular , Ácidos Nucleicos/metabolismo , Filogenia , Ribonucleasa H/metabolismoRESUMEN
In previous work with thermally robust salts [Cassity et al., Phys. Chem. Chem. Phys., 2017, 19, 31560] it was noted that an increase in the dipole moment of the cation generally led to a decrease in the melting point. Molecular dynamics simulations of the liquid state revealed that an increased dipole moment reduces cation-cation repulsions through dipole-dipole alignment. This was believed to reduce the liquid phase enthalpy, which would tend to lower the melting point of the IL. In this work we further test this principle by replacing hydrogen atoms with fluorine atoms at selected positions within the cation. This allows us to alter the electrostatics of the cation without substantially affecting the sterics. Furthermore, the strength of the dipole moment can be controlled by choosing different positions within the cation for replacement. We studied variants of four different parent cations paired with bistriflimide and determined their melting points, and enthalpies and entropies of fusion through DSC experiments. The decreases in the melting point were determined to be enthalpically driven. We found that the dipole moment of the cation, as determined by quantum chemical calculations, is inversely correlated with the melting point of the given compound. Molecular dynamics simulations of the crystalline and solid states of two isomers showed differences in their enthalpies of fusion that closely matched those seen experimentally. Moreover, this reduction in the enthalpy of fusion was determined to be caused by an increase in the enthalpy of the crystalline state. We provide evidence that dipole-dipole interactions between cations leads to the formation of cationic domains in the crystalline state. These cationic associations partially block favourable cation-anion interactions, which are recovered upon melting. If, however, the dipole-dipole interactions between cations is too strong they have a tendency to form glasses. This study provides a design rule for lowering the melting point of structurally similar ILs by altering their dipole moment.
RESUMEN
RNA-mediated transcriptional silencing, in plants known as RNA-directed DNA methylation (RdDM), is a conserved process where small interfering RNA (siRNA) and long non-coding RNA (lncRNA) help establish repressive chromatin modifications. This process represses transposons and affects the expression of protein-coding genes. We found that in Arabidopsis thaliana AGO4 binding sites are often located distant from genes differentially expressed in ago4. Using Hi-C to compare interactions between genotypes, we show that RdDM-targeted loci have the potential to engage in chromosomal interactions, but these interactions are inhibited in wild-type conditions. In mutants defective in RdDM, the frequency of chromosomal interactions at RdDM targets is increased. This includes increased frequency of interactions between Pol V methylated sites and distal genes that are repressed by RdDM. We propose a model, where RdDM prevents the formation of chromosomal interactions between genes and their distant regulatory elements.
Asunto(s)
Arabidopsis/genética , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , ARN Largo no Codificante/genética , ARN Interferente Pequeño/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Epistasis GenéticaRESUMEN
Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Citosina/metabolismo , Metilación de ADN , ARN Polimerasas Dirigidas por ADN , Genoma de Planta , ARN Interferente Pequeño/metabolismo , Secuencias de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , ARN Interferente Pequeño/genéticaRESUMEN
BIR1 is a receptor-like kinase that functions as a negative regulator of basal immunity and cell death in Arabidopsis. Using Arabidopsis thaliana and Tobacco rattle virus (TRV), we investigate the antiviral role of BIR1, the molecular mechanisms of BIR1 gene expression regulation during viral infections, and the effects of BIR1 overexpression on plant immunity and development. We found that SA acts as a signal molecule for BIR1 activation during infection. Inactivating mutations of BIR1 in the bir1-1 mutant cause strong antiviral resistance independently of constitutive cell death or SA defense priming. BIR1 overexpression leads to severe developmental defects, cell death and premature death, which correlate with the constitutive activation of plant immune responses. Our findings suggest that BIR1 acts as a negative regulator of antiviral defense in plants, and indicate that RNA silencing contributes, alone or in conjunction with other regulatory mechanisms, to define a threshold expression for proper BIR1 function beyond which an autoimmune response may occur. This work provides novel mechanistic insights into the regulation of BIR1 homeostasis that may be common for other plant immune components.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/virología , Inmunidad de la Planta/genética , Virus de Plantas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transcripción Genética , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Metilación de ADN/genética , Silenciador del Gen , Mutación/genética , Fenotipo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Proteínas Represoras/metabolismo , Ácido Salicílico/farmacología , Regulación hacia Arriba/genéticaRESUMEN
Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs.
Asunto(s)
Bryopsida/genética , Plantas/genética , ARN Interferente Pequeño/genética , ARN Pequeño no Traducido/genética , Secuencia de Aminoácidos , Secuencia de Bases , Vías Biosintéticas/genética , Evolución Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Genes de Plantas/genética , Heterocromatina/genética , MicroARNs/genética , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Filogenia , Plantas/clasificación , ARN Interferente Pequeño/clasificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The remarkable properties of bone derive from a highly organized arrangement of coaligned nanometer-scale apatite platelets within a fibrillar collagen matrix. The origin of this arrangement is poorly understood and the crystal structures of hydroxyapatite (HAP) and the nonmineralized collagen fibrils alone do not provide an explanation. Moreover, little is known about collagen-apatite interaction energies, which should strongly influence both the molecular-scale organization and the resulting mechanical properties of the composite. We investigated collagen-mineral interactions by combining dynamic force spectroscopy (DFS) measurements of binding energies with molecular dynamics (MD) simulations of binding and atomic force microscopy (AFM) observations of collagen adsorption on single crystals of calcium phosphate for four mineral phases of potential importance in bone formation. In all cases, we observe a strong preferential orientation of collagen binding, but comparison between the observed orientations and transmission electron microscopy (TEM) analyses of native tissues shows that only calcium-deficient apatite (CDAP) provides an interface with collagen that is consistent with both. MD simulations predict preferred collagen orientations that agree with observations, and results from both MD and DFS reveal large values for the binding energy due to multiple binding sites. These findings reconcile apparent contradictions inherent in a hydroxyapatite or carbonated apatite (CAP) model of bone mineral and provide an energetic rationale for the molecular-scale organization of bone.
Asunto(s)
Huesos/química , Huesos/metabolismo , Animales , Sitios de Unión , Huesos/ultraestructura , Bovinos , Dentina/química , Dentina/metabolismo , Dentina/ultraestructura , Durapatita/química , Durapatita/metabolismo , Metabolismo Energético , Colágenos Fibrilares/química , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/ultraestructura , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Modelos Moleculares , Simulación de Dinámica Molecular , Nanoestructuras/química , Nanoestructuras/ultraestructura , RatasRESUMEN
The Arabidopsis histone deacetylase HDA6 is required to silence transgenes, transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar dominance in genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S rRNA gene variants that is normally inactivated during development fails to be silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription occurs throughout the intergenic spacers. The resulting sense and antisense spacer transcripts facilitate a massive overproduction of siRNAs that, in turn, direct de novo cytosine methylation of corresponding gene sequences. However, the resulting de novo DNA methylation fails to suppress Pol I or Pol II transcription in the absence of HDA6 activity; instead, euchromatic histone modifications typical of active genes accumulate. Collectively, the data reveal a futile cycle of unregulated transcription, siRNA production, and siRNA-directed DNA methylation in the absence of HDA6-mediated histone deacetylation. We propose that spurious Pol II transcription throughout the intergenic spacers in hda6 mutants, combined with losses of histone deacetylase activity and/or maintenance DNA methylation, eliminates repressive chromatin modifications needed for developmental rRNA gene dosage control.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Citosina/metabolismo , ADN Polimerasa II/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de ARNr/genética , Histona Desacetilasas/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Arabidopsis/genética , ADN Intergénico/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Histona Desacetilasas/genética , Histonas/metabolismo , Metilación , MutaciónRESUMEN
A family of mesothermal ionic liquids comprised of tetraarylphosphonium cations and the bis(trifluoromethanesulfonyl)amidate anion are shown to be materials of exceptional thermal stability, enduring (without decomposition) heating in air at 300 °C for three months. It is further established that three specific structural elements - phenoxy, phenacyl, and phenyl sulfonyl - can be present in the cation structures without compromising their thermal stability, and that their incorporation has specific impacts on the melting points of the salts. Most importantly, it is shown that the ability of such a structural component to lower a salt melting point is tied to its ability to lower cation-cation repulsions in the material.