RESUMO
To gain a deeper insight into cellular processes such as transcription and translation, one needs to uncover the mechanisms controlling the configurational changes of nucleic acids. As a step toward this aim, we present here a mesoscopic-level computational model that provides a new window into nucleic acid dynamics. We model a single-stranded nucleic as a polymer chain whose monomers are the nucleosides. Each monomer comprises a bead representing the sugar molecule and a pin representing the base. The bead-pin complex can rotate about the backbone of the chain. We consider pairwise stacking and hydrogen-bonding interactions. We use a modified Monte Carlo dynamics that splits the dynamics into translational bead motion and rotational pin motion. By performing a number of tests, we first show that our model is physically sound. We then focus on a study of the kinetics of a DNA hairpin--a single-stranded molecule comprising two complementary segments joined by a noncomplementary loop--studied experimentally. We find that results from our simulations agree with experimental observations, demonstrating that our model is a suitable tool for the investigation of the hybridization of single strands.
Assuntos
DNA/química , Hibridização In Situ/métodos , Modelos Químicos , Modelos Moleculares , Nucleotídeos/química , RNA/química , Sítios de Ligação , Simulação por Computador , DNA/análise , Cinética , Movimento (Física) , Conformação de Ácido Nucleico , Nucleotídeos/análise , RNA/análise , Relação Estrutura-AtividadeRESUMO
Gene-regulatory complexes often require that pairs of DNA-bound proteins interact by looping-out short (often approximately 100-bp) stretches of DNA. The loops can vary in detailed length and sequence and, thus, in total helical twist, which radically alters their geometry. How this variability is accommodated structurally is not known. Here we show that the inherent twistability of 89- to 105-bp DNA circles exceeds theoretical expectation by up to 400-fold. These results can be explained only by greatly enhanced DNA flexibility, not by permanent bends. They invalidate the use of classic theories of flexibility for understanding sharp DNA looping but support predictions of two recent theories. Our findings imply an active role for DNA flexibility in loop formation and suggest that variability in the detailed helical twist of regulatory loops is accommodated naturally by the inherent twistability of the DNA.
Assuntos
DNA/química , Proteínas/química , Regulação da Expressão Gênica , Conformação de Ácido NucleicoRESUMO
DNA sequence is an important determinant of the positioning, stability, and activity of nucleosomes, yet the molecular basis of these effects remains elusive. A "consensus DNA sequence" for nucleosome positioning has not been reported and, while certain DNA sequence preferences or motifs for nucleosome positioning have been discovered, how they function is not known. Here, we report that an unexpected observation concerning the reassembly of nucleosomes during salt gradient dialysis has allowed a breakthrough in our efforts to identify the nucleosomal locations of the DNA sequence motifs that dominate histone-DNA interactions and nucleosome positioning. We conclude that a previous selection experiment for high-affinity, nucleosome-forming DNA sequences exerted selective pressure chiefly on the central stretch of the nucleosomal DNA. This observation implies that algorithms for aligning the selected DNA sequences should seek to optimize the alignment over much less than the full 147 bp of nucleosomal DNA. A new alignment calculation implemented these ideas and successfully aligned 19 of the 41 sequences in a non-redundant database of selected high-affinity, nucleosome-positioning sequences. The resulting alignment reveals strong conservation of several stretches within a central 71 bp of the nucleosomal DNA. The alignment further reveals an inherent palindromic symmetry in the selected DNAs; it makes testable predictions of nucleosome positioning on the aligned sequences and for the creation of new positioning sequences, both of which are upheld experimentally; and it suggests new signals that may be important in translational nucleosome positioning.
Assuntos
Sequência de Bases , DNA/genética , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Algoritmos , Composição de Bases , Dados de Sequência Molecular , Alinhamento de Sequência , TermodinâmicaRESUMO
Nucleosome positioning DNA sequences are of increasing interest because of their proposed roles in gene regulation and other chromosome functions in vivo, and because they have revealed new insights into the sequence-dependent structures and mechanics of DNA itself. Here, we describe methods to quantify the relative affinities of histone-DNA interactions in nucleosomes, i.e., the nucleosome positioning power of differing DNA sequences. We review methods developed by others and then discuss in detail our own approach to measurement of histone-DNA interaction free energies. Compared to earlier methods, our dialysis-based approach reduces the possibility that non-equilibrium or irreproducible results could be obtained. It facilitates a direct comparison of free energies for many sequences at the same time and it allows analysis of DNAs having a wide range of relative affinities.
Assuntos
DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Termodinâmica , Animais , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Cinética , CamundongosAssuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Drosophila/metabolismo , Polarização de Fluorescência/instrumentação , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Animais , DNA/metabolismo , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Fluoresceína , Polarização de Fluorescência/métodos , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
Despite decades of study on nucleosomes, there has been no experimental determination of the free energy of association between histones and DNA. Instead, only the relative free energy of association of the histone octamer for differing DNA sequences has been available. Recently, a method was developed based on quantitative analysis of nucleosome dissociation in dilution experiments that provides a simple practical measure of nucleosome stability. Solution conditions were found in which nucleosome dissociation driven by dilution fit well to a simple model involving a noncooperative nucleosome assembly/disassembly equilibrium, suggesting that this approach might allow absolute equilibrium affinity of the histone octamer for DNA to be measured. Here, we show that the nucleosome assembly/disassembly process is not strictly reversible in these solution conditions, implying that equilibrium affinities cannot be obtained from these measurements. Increases in [NaCl] or temperature, commonly employed to suppress kinetic bottlenecks in nucleosome assembly, lead to cooperative behavior that cannot be interpreted with the simple assembly/disassembly equilibrium model. We conclude that the dilution experiments provide useful measures of kinetic but not equilibrium stability. Kinetic stability is of practical importance: it may govern nucleosome function in vivo, and it may (but need not) parallel absolute thermodynamic stability.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Animais , Soluções Tampão , Galinhas , Detergentes , Glicerol , Glicogênio , Cinética , Nucleossomos/química , Nucleossomos/metabolismo , Octoxinol , Polietilenoglicóis , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Soluções , Termodinâmica , Trometamina , Xenopus laevisRESUMO
Intrinsic nucleosome dynamics termed "site exposure" provides spontaneous and cooperative access to buried regions of nucleosomal DNA in vitro. Two different mechanisms for site exposure have been proposed, one based on nucleosome translocation, the other on dynamic nucleosome conformational changes in which a stretch of the nucleosomal DNA is transiently released off the histone surface. Here we report on three experiments that distinguish between these mechanisms. One experiment investigates the effects on the accessibilities of restriction enzyme target sites inside nucleosomes when extra DNA (onto which the nucleosome may move at low energetic cost) is appended onto one end. The other two experiments test directly for nucleosome mobility under the conditions used to probe accessibility to restriction enzymes: one on a selected nonnatural nucleosome positioning sequence, the other on the well-studied 5S rRNA gene nucleosome positioning sequence. We find from all three assays that restriction enzymes gain access to sites throughout the entire length of the nucleosomal DNA without contribution from nucleosome translocation. We conclude that site exposure in nucleosomes in vitro occurs via a nucleosome conformational change that leads to transient release of a stretch of DNA from the histone surface, most likely involving progressive uncoiling from an end. Recapture at a distal site along DNA that has partially uncoiled would result in looped structures which are believed to contribute to RNA polymerase elongation and may contribute to spontaneous or ATP-driven nucleosome mobility. Transient open states may facilitate the initial entry of transcription factors and enzymes in vivo.
Assuntos
DNA/metabolismo , Nucleossomos/metabolismo , Proteínas/metabolismo , Animais , Transporte Biológico , Galinhas , DNA Ribossômico/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Desvio de Mobilidade Eletroforética , Camundongos , RNA Ribossômico 5S , Moldes GenéticosRESUMO
Polypurine tracts are important elements of eukaryotic promoters. They are believed to somehow destabilize chromatin, but the mechanism of their action is not known. We show that incorporating an A(16) element at an end of the nucleosomal DNA and further inward destabilizes histone-DNA interactions by 0.1 +/- 0.03 and 0.35 +/- 0.04 kcal mol(-1), respectively, and is accompanied by 1.5- +/- 0.1-fold and 1.7- +/- 0.1-fold increases in position-averaged equilibrium accessibility of nucleosomal DNA target sites. These effects are comparable in magnitude to effects of A(16) elements that correlate with transcription in vivo, suggesting that our system may capture most of their physiological role. These results point to two distinct but interrelated models for the mechanism of action of polypurine tract promoter elements in vivo. Given a nucleosome positioned over a promoter region, the presence of a polypurine tract in that nucleosome's DNA decreases the stability of the DNA wrapping, increasing the equilibrium accessibility of other DNA target sites buried inside that nucleosome. Alternatively (if nucleosomes are freely mobile), the presence of a polypurine tract provides a free energy bias for the nucleosome to move to alternative locations, thereby changing the equilibrium accessibilities of other nearby DNA target sites.
Assuntos
DNA/metabolismo , Nucleossomos/metabolismo , Poli dA-dT/genética , Regiões Promotoras Genéticas , Sítios de Ligação , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Cinética , Moldes GenéticosRESUMO
We consider how beads can diffuse along a chain that wraps them, without becoming displaced from the chain; our proposed mechanism is analogous to the reptation of "stored length" in more familiar situations of polymer dynamics. The problem arises in the case of globular aggregates of proteins (histones) that are wound by DNA in the chromosomes of plants and animals; these beads (nucleosomes) are multiply wrapped and yet are able to reposition themselves over long distances, while remaining bound by the DNA chain.
Assuntos
DNA/química , Modelos Biológicos , Nucleossomos/química , Animais , DNA/fisiologia , DNA de Plantas/química , DNA de Plantas/fisiologia , DNA Super-Helicoidal/química , DNA Super-Helicoidal/fisiologia , Histonas/química , Histonas/fisiologia , Conformação de Ácido Nucleico , Nucleossomos/fisiologiaRESUMO
Posttranslational acetylation of the conserved core histone N-terminal tail domains is linked to gene activation, but the molecular mechanisms involved are not known. In an earlier study we showed that removing the tail domains altogether by trypsin proteolysis (which leaves nucleosomes nevertheless intact) leads to 1.5 to 14-fold increases in the dynamic equilibrium accessibility of nucleosomal DNA target sites. These observations suggested that, by modestly increasing the equilibrium accessibility of buried DNA target sites, histone acetylation could result in an increased occupancy by regulatory proteins, ultimately increasing the probability of transcription initiation. Here, we extend these observations to a more natural system involving intact but hyperacetylated nucleosomes. We find that histone hyperacetylation leads to 1.1 to 1.8-fold increases in position-dependent equilibrium constants for exposure of nucleosomal DNA target sites, with an average increase of 1.4(+/-0.1)-fold. The mechanistic and biological implications of these results are discussed.
Assuntos
DNA/metabolismo , Histonas/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Acetilação , Acetiltransferases/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Galinhas , DNA/química , DNA/genética , Enzimas de Restrição do DNA/metabolismo , Eritrócitos , Regulação da Expressão Gênica , Células HeLa , Histona Acetiltransferases , Histonas/química , Histonas/genética , Humanos , Cinética , Ensaios de Proteção de Nucleases , Nucleossomos/genética , Moldes Genéticos , Termodinâmica , Ativação TranscricionalRESUMO
The N-terminal tail domains of the core histones play important roles in gene regulation, but the exact mechanisms through which they act are not known. Recent studies suggest that the tail domains may influence the ability of RNA polymerase to elongate through the nucleosomal DNA and, thus, that posttranslational modification of the tail domains may provide a control point for gene regulation through effects on the elongation rate. We take advantage of an experimental system that uses bacteriophage T7 RNA polymerase as a probe for aspects of nucleosome transcription that are dominated by the properties of nucleosomes themselves. With this system, experiments can analyze the synchronous, real-time, single-passage transcription on the nucleosomal template. Here, we use this system to directly test the hypothesis that the tail domains may influence the "elongatability" of nucleosomal DNA and to identify which of the tail domains may contribute to this. The results show that the tail domains strongly influence the rate of elongation and suggest that the effect is dominated by the N-terminal domains of the (H3-H4)(2) tetramer. They further imply that tail-mediated octamer transfer is not essential for elongation through the nucleosome. Acetylation of the tail domains leads to effects on elongation that are similar to those arising from complete removal of the tail domains.
Assuntos
Cromatina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Pegada de DNA , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas ViraisRESUMO
Archaeal histones and the eucaryal (eucaryotic) nucleosome core histones have almost identical histone folds. Here, we show that DNA molecules selectively incorporated by rHMfB (recombinant archaeal histone B from Methanothermus fervidus) into archaeal nucleosomes from a mixture of approximately 10(14) random sequence molecules contain sequence motifs shown previously to direct eucaryal nucleosome positioning. The dinucleotides GC, AA (=TT) and TA are repeated at approximately 10 bp intervals, with the GC harmonic displaced approximately 5 bp from the AA and TA harmonics [(GCN(3)AA or TA)(n)]. AT and CG were not strongly selected, indicating that TA not equalAT and GC not equalCG in terms of facilitating archaeal nucleosome assembly. The selected molecules have affinities for rHMfB ranging from approximately 9 to 18-fold higher than the level of affinity of the starting population, and direct the positioned assembly of archaeal nucleosomes. Fourier-transform analyses have revealed that AA dinucleotides are much enriched at approximately 10. 1 bp intervals, the helical repeat of DNA wrapped around a nucleosome, in the genomes of Eucarya and the histone-containing Euryarchaeota, but not in the genomes of Bacteria and Crenarchaeota, procaryotes that do not have histones. Facilitating histone packaging of genomic DNA has apparently therefore imposed constraints on genome sequence evolution, and since archaeal histones have no structure in addition to the histone fold, these constraints must result predominantly from histone fold-DNA contacts. Based on the three-domain universal phylogeny, histones and histone-dependent genome sequence evolution most likely evolved after the bacterial-archaeal divergence but before the archaeal-eucaryal divergence, and were subsequently lost in the Crenarchaeota. However, with lateral gene transfer, the first histone fold could alternatively have evolved after the archaeal-eucaryal divergence, early in either the euryarchaeal or eucaryal lineages.
Assuntos
Archaea , DNA/genética , Evolução Molecular , Genoma Bacteriano , Histonas/metabolismo , Nucleossomos/genética , Archaea/química , Archaea/citologia , Archaea/genética , Sequência de Bases , Clonagem Molecular , DNA/química , DNA/metabolismo , Pegada de DNA , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Repetições de Dinucleotídeos/genética , Células Eucarióticas , Análise de Fourier , Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Histonas/química , Nuclease do Micrococo/metabolismo , Dados de Sequência Molecular , Nucleossomos/química , Nucleossomos/metabolismo , FilogeniaRESUMO
RosR is a transcriptional regulator important for determining cell-surface characteristics and nodulation competitiveness in Rhizobium etli CE3. We identified a 15-kb region that contains genes with similarity to members of the virB, virC, virG, and virE operons of Agrobacterium tumefaciens and demonstrated that RosR directly regulates one operon in this region. These genes were located on plasmid pa of R. etli CE3, which is self-transmissible between R. etli and A. tumefaciens.
Assuntos
Genes Bacterianos , Plasmídeos , Rhizobium/genética , Virulência/genética , Sequência de Bases , Southern Blotting , Primers do DNA , Eletroforese em Gel de PoliacrilamidaRESUMO
Mutations in palmitoyl-protein thioesterase 1 (PPT1), a lysosomal enzyme that removes fatty acyl groups from cysteine residues in modified proteins, cause the fatal inherited neurodegenerative disorder infantile neuronal ceroid lipofuscinosis. The accumulation of undigested substrates leads to the formation of neuronal storage bodies that are associated with the clinical symptoms. Less severe forms of PPT1 deficiency have been found recently that are caused by a distinct set of PPT1 mutations, some of which retain a small amount of thioesterase activity. We have determined the crystal structure of PPT1 with and without bound palmitate by using multiwavelength anomalous diffraction phasing. The structure reveals an alpha/beta-hydrolase fold with a catalytic triad composed of Ser115-His289-Asp233 and provides insights into the structural basis for the phenotypes associated with PPT1 mutations.
Assuntos
Lipofuscinoses Ceroides Neuronais/enzimologia , Lipofuscinoses Ceroides Neuronais/genética , Tioléster Hidrolases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Cristalografia por Raios X , Humanos , Lactente , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Tioléster Hidrolases/deficiência , Tioléster Hidrolases/genéticaRESUMO
The N and C-terminal tail domains of the core histones play important roles in gene regulation, but the mechanisms through which they act are not known. These tail domains are highly positively charged and are the sites of numerous post-translational modifications, including many sites for lysine acetylation. Nucleosomes in which these tail domains have been removed by trypsin remain otherwise intact, and are used by many laboratories as a model system for highly acetylated nucleosomes. Here, we test the hypothesis that one role of the tail domains is to directly regulate the accessibility of nucleosomal DNA to other DNA-binding proteins. Three assays are used: equilibrium binding by a site-specific, DNA-binding protein, and dynamic accessibility to restriction enzymes or to a non-specific exonuclease. The effects of removal of the tail domains as monitored by each of these assays can be understood within the framework of the site exposure model for the dynamic equilibrium accessibility of target sites located within the nucleosomal DNA. Removal of the tail domains leads to a 1.5 to 14-fold increase in position-dependent equilibrium constants for site exposure. The smallness of the effect weighs against models for gene activation in which histone acetylation is a mandatory initial event, required to facilitate subsequent access of regulatory proteins to nucleosomal DNA target sites. Alternative roles for histone acetylation in gene regulation are discussed.
Assuntos
DNA/metabolismo , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Acetilação , Animais , Sítios de Ligação , Galinhas , DNA/química , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eritrócitos , Exodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Histonas/isolamento & purificação , Cinética , Modelos Genéticos , Nucleossomos/genética , Ligação Proteica , Proteínas de Saccharomyces cerevisiae , Termodinâmica , Titulometria , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
We have previously shown that nucleosomes are conformationally dynamic: DNA sequences that in the time-average are buried inside nucleosomes are nevertheless transiently accessible, even to large proteins (or any other macromolecule). We refer to this dynamic behavior as "site exposure". Here we show that: (i) the equilibrium constants describing this dynamic site exposure decrease progressively from either end of the nucleosomal DNA in toward the middle; and (ii) these position-dependent equilibrium constants are strongly dependent on the nucleosomal DNA sequence. The progressive decrease in equilibrium constant with distance inside the nucleosome supports the hypothesis that access to sites internal to a nucleosome is provided by progressive (transient) release of DNA from the octamer surface, starting from one end of the nucleosomal DNA. The dependence on genomic DNA sequence implies that a specific genomic DNA sequence could be a major determinant of target site occupancies achieved by regulatory proteins in vivo, by either governing the time-averaged accessibility for a given nucleosome position, or biasing the time-averaged positioning (of mobile nucleosomes), which in turn is a major determinant of site accessibility.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Nucleossomos/metabolismo , DNA/genética , Enzimas de Restrição do DNA/química , Metabolismo Energético , Regulação da Expressão Gênica , Cinética , Conformação de Ácido Nucleico , Nucleossomos/química , Sequências Reguladoras de Ácido NucleicoAssuntos
Enzimas de Restrição do DNA , DNA/química , Nucleossomos/genética , Nucleossomos/ultraestrutura , Mapeamento por Restrição/métodos , Animais , Galinhas , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Eritrócitos/ultraestrutura , Histonas/química , Histonas/metabolismo , Cinética , Modelos Químicos , Moldes GenéticosRESUMO
Our laboratories recently completed SELEX experiments to isolate DNA sequences that most-strongly favor or disfavor nucleosome formation and positioning, from the entire mouse genome or from even more diverse pools of chemically synthetic random sequence DNA. Here we directly compare these selected natural and non-natural sequences. We find that the strongest natural positioning sequences have affinities for histone binding and nucleosome formation that are sixfold or more lower than those possessed by many of the selected non-natural sequences. We conclude that even the highest-affinity sequence regions of eukaryotic genomes are not evolved for the highest affinity or nucleosome positioning power. Fourier transform calculations on the selected natural sequences reveal a special significance for nucleosome positioning of a motif consisting of approximately 10 bp periodic placement of TA dinucleotide steps. Contributions to histone binding and nucleosome formation from periodic TA steps are more significant than those from other periodic steps such as AA (=TT), CC (=GG) and more important than those from the other YR steps (CA (=TG) and CG), which are reported to have greater conformational flexibility in protein-DNA complexes even than TA. We report the development of improved procedures for measuring the free energies of even stronger positioning sequences that may be isolated in the future, and show that when the favorable free energy of histone-DNA interactions becomes sufficiently large, measurements based on the widely used exchange method become unreliable.