RESUMEN
In the plant sciences, results of laboratory studies often do not translate well to the field. To help close this lab-field gap, we developed a strategy for studying the wiring of plant traits directly in the field, based on molecular profiling and phenotyping of individual plants. Here, we use this single-plant omics strategy on winter-type Brassica napus (rapeseed). We investigate to what extent early and late phenotypes of field-grown rapeseed plants can be predicted from their autumnal leaf gene expression, and find that autumnal leaf gene expression not only has substantial predictive power for autumnal leaf phenotypes but also for final yield phenotypes in spring. Many of the top predictor genes are linked to developmental processes known to occur in autumn in winter-type B. napus accessions, such as the juvenile-to-adult and vegetative-to-reproductive phase transitions, indicating that the yield potential of winter-type B. napus is influenced by autumnal development. Our results show that single-plant omics can be used to identify genes and processes influencing crop yield in the field.
Asunto(s)
Brassica napus , Brassica napus/genética , Hojas de la Planta/genética , Fenotipo , Expresión GénicaRESUMEN
DNA supercoiling acts as a global transcriptional regulator in bacteria, but the promoter sequence or structural determinants controlling its effect remain unclear. It was previously proposed to modulate the torsional angle between the -10 and -35 hexamers, and thereby regulate the formation of the closed-complex depending on the length of the 'spacer' between them. Here, we develop a thermodynamic model of this notion based on DNA elasticity, providing quantitative and parameter-free predictions of the relative activation of promoters containing a short versus long spacer when the DNA supercoiling level is varied. The model is tested through an analysis of in vitro and in vivo expression assays of mutant promoters with variable spacer lengths, confirming its accuracy for spacers ranging from 15 to 19 nucleotides, except those of 16 nucleotides where other regulatory mechanisms likely overcome the effect of this specific step. An analysis at the whole-genome scale in Escherichia coli then demonstrates a significant effect of the spacer length on the genomic expression after transient or inheritable superhelical variations, validating the model's predictions. Altogether, this study shows an example of mechanical constraints associated to promoter binding by RNA Polymerase underpinning a basal and global regulatory mechanism.
Asunto(s)
ADN Bacteriano , ADN Superhelicoidal , Regiones Promotoras Genéticas , Transcripción Genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal/genética , Escherichia coli/genética , Escherichia coli/metabolismo , NucleótidosRESUMEN
DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by DNA gyrase inhibition were categorised as 'supercoiling-sensitive'. Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after DNA gyrase or topoisomerase I inhibition.
Asunto(s)
Girasa de ADN , ADN-Topoisomerasas de Tipo I , Girasa de ADN/genética , Girasa de ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Antibacterianos/farmacologíaRESUMEN
The catabolism of pectin from plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce massive pectinolytic activity during the maceration phase. Previous studies identified the role of a positive feedback loop specific to the pectin-degradation pathway, whereas the precise signals controlling the dynamics of pectate lyase expression were unclear. Here, we show that the latter is controlled by a metabolic switch involving both glucose and pectin. We measured the HPLC concentration profiles of the key metabolites related to these two sources of carbon, cAMP and 2-keto-3-deoxygluconate, and developed a dynamic and quantitative model of the process integrating the associated regulators, cAMP receptor protein and KdgR. The model describes the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights that their activity is controlled by a mechanism of carbon catabolite repression, which directly controls the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitatively different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, justifying their evolutionary conservation as separate genes in this species.
Asunto(s)
Represión Catabólica , Dickeya , Pectinas , Proteínas Bacterianas/metabolismo , Dickeya/metabolismo , Digestión , Enterobacteriaceae/metabolismo , Regulación Bacteriana de la Expresión Génica , Pectinas/metabolismo , Polisacárido Liasas/químicaRESUMEN
Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαß heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a 'transcriptional domainin' protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.
Asunto(s)
Proteínas Bacterianas/fisiología , Dickeya/patogenicidad , Regulación Bacteriana de la Expresión Génica , Factores de Integración del Huésped/fisiología , Proteínas Bacterianas/genética , Sitios de Unión , Celulasa/biosíntesis , Celulasa/genética , Cichorium intybus/microbiología , ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Dickeya/genética , Dickeya/fisiología , Dimerización , Estudios de Asociación Genética , Factores de Integración del Huésped/química , Factores de Integración del Huésped/genética , Movimiento (Física) , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/genética , Plásmidos , Poligalacturonasa/biosíntesis , Poligalacturonasa/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Sideróforos/biosíntesis , Sideróforos/genética , Transcripción Genética/genética , Transcriptoma , Virulencia/genéticaRESUMEN
This study investigated modifications to the ubiquitin proteasome system (UPS) in a mouse model of type 2 diabetes mellitus (T2DM) and their relationship to heart complications. db/db mice heart tissues were compared with WT mice tissues using RNA sequencing, qRT-PCR, and protein analysis to identify cardiac UPS modifications associated with diabetes. The findings unveiled a distinctive gene profile in the hearts of db/db mice with decreased levels of nppb mRNA and increased levels of Myh7, indicating potential cardiac dysfunction. The mRNA levels of USP18 (deubiquitinating enzyme), PSMB8, and PSMB9 (proteasome ß-subunits) were down-regulated in db/db mice, while the mRNA levels of RNF167 (E3 ligase) were increased. Corresponding LMP2 and LMP7 proteins were down-regulated in db/db mice, and RNF167 was elevated in Adult diabetic mice. The reduced expression of LMP2 and LMP7, along with increased RNF167 expression, may contribute to the future cardiac deterioration commonly observed in diabetes. This study enhances our understanding of UPS imbalances in the hearts of diabetic mice and raises questions about the interplay between the UPS and other cellular processes, such as autophagy. Further exploration in this area could provide valuable insights into the mechanisms underlying diabetic heart complications and potential therapeutic targets.
Asunto(s)
Complicaciones de la Diabetes , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Cardiomiopatías Diabéticas , Ratones , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/metabolismo , Complicaciones de la Diabetes/complicaciones , ARN Mensajero/genéticaRESUMEN
DNA supercoiling, the level of under- or overwinding of the DNA polymer around itself, is widely recognized as an ancestral regulation mechanism of gene expression in bacteria. Higher levels of negative supercoiling facilitate the opening of the DNA double helix at gene promoters and thereby increase gene transcription rates. Different levels of supercoiling have been measured in bacteria exposed to different environments, leading to the hypothesis that variations in supercoiling could be a response to changes in the environment. Moreover, DNA transcription has been shown to generate local variations in the supercoiling level and, therefore, to impact the transcription rate of neighboring genes. In this work, we study the coupled dynamics of DNA supercoiling and transcription at the genome scale. We implement a genome-wide model of gene expression based on the transcription-supercoiling coupling. We show that, in this model, a simple change in global DNA supercoiling is sufficient to trigger differentiated responses in gene expression levels via the transcription-supercoiling coupling. Then, studying our model in the light of evolution, we demonstrate that this non-linear response to different environments, mediated by the transcription-supercoiling coupling, can serve as the basis for the evolution of specialized phenotypes.
Asunto(s)
ADN Superhelicoidal , Transcripción Genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Regiones Promotoras Genéticas , ADNRESUMEN
SUMMARY: Transcription and DNA supercoiling are involved in a complex, dynamical and non-linear coupling that results from the basal interaction between DNA and RNA polymerase. We present the first software to simulate this coupling, applicable to a wide range of bacterial organisms. TwisTranscripT allows quantifying its contribution in global transcriptional regulation, and provides a mechanistic basis for the widely observed, evolutionarily conserved and currently unexplained co-regulation of adjacent operons that might play an important role in genome evolution. AVAILABILITY AND IMPLEMENTATION: TwisTranscripT is freely available at https://github.com/sammeyer2017/TwisTranscripT. It is implemented in Python3 and supported on MacOS X, Linux and Windows.
Asunto(s)
Operón , Programas Informáticos , Bacterias , Simulación por ComputadorRESUMEN
Most of our current knowledge on plant molecular biology is based on experiments in controlled laboratory environments. However, translating this knowledge from the laboratory to the field is often not straightforward, in part because field growth conditions are very different from laboratory conditions. Here, we test a new experimental design to unravel the molecular wiring of plants and study gene-phenotype relationships directly in the field. We molecularly profiled a set of individual maize plants of the same inbred background grown in the same field and used the resulting data to predict the phenotypes of individual plants and the function of maize genes. We show that the field transcriptomes of individual plants contain as much information on maize gene function as traditional laboratory-generated transcriptomes of pooled plant samples subject to controlled perturbations. Moreover, we show that field-generated transcriptome and metabolome data can be used to quantitatively predict individual plant phenotypes. Our results show that profiling individual plants in the field is a promising experimental design that could help narrow the lab-field gap.
Asunto(s)
Genes de Plantas , Genómica , Zea mays/genética , Análisis por Conglomerados , Análisis de Datos , Bases de Datos Genéticas , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Metaboloma/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Estrés Fisiológico/genética , Transcriptoma/genética , Zea mays/crecimiento & desarrolloRESUMEN
A rapid and sensitive High Performance Liquid Chromatography (HPLC) method with photometric and fluorescence detection is developed for routine analysis of 2-Keto-3-deoxy-gluconate (KDG), a catabolite product of pectin and alginate. These polysaccharides are primary-based compounds for biofuel production and for generation of high-value-added products. HPLC is performed, after derivatization of the 2-oxo-acid groups of the metabolite with o-phenylenediamine (oPD), using a linear gradient of trifluoroacetic acid and acetonitrile. Quantification is accomplished with an internal standard method. The gradient is optimized to distinguish KDG from its close structural analogues such as 5-keto-4-deoxyuronate (DKI) and 2,5-diketo-3-deoxygluconate (DKII). The proposed method is simple, highly sensitive and accurate for time course analysis of pectin or alginate degradation.
Asunto(s)
Alginatos/metabolismo , Dickeya/metabolismo , Gluconatos , Pectinas/metabolismo , Gluconatos/química , Gluconatos/aislamiento & purificación , Gluconatos/metabolismoRESUMEN
Small non-coding RNAs (sRNAs) regulate numerous cellular processes in all domains of life. Several approaches have been developed to identify them from RNA-seq data, which are efficient for eukaryotic sRNAs but remain inaccurate for the longer and highly structured bacterial sRNAs. We present APERO, a new algorithm to detect small transcripts from paired-end bacterial RNA-seq data. In contrast to previous approaches that start from the read coverage distribution, APERO analyzes boundaries of individual sequenced fragments to infer the 5' and 3' ends of all transcripts. Since sRNAs are about the same size as individual fragments (50-350 nucleotides), this algorithm provides a significantly higher accuracy and robustness, e.g., with respect to spontaneous internal breaking sites. To demonstrate this improvement, we develop a comparative assessment on datasets from Escherichia coli and Salmonella enterica, based on experimentally validated sRNAs. We also identify the small transcript repertoire of Dickeya dadantii including putative intergenic RNAs, 5' UTR or 3' UTR-derived RNA products and antisense RNAs. Comparisons to annotations as well as RACE-PCR experimental data confirm the precision of the detected transcripts. Altogether, APERO outperforms all existing methods in terms of sRNA detection and boundary precision, which is crucial for comprehensive genome annotations. It is freely available as an open source R package on https://github.com/Simon-Leonard/APERO.
Asunto(s)
Algoritmos , Escherichia coli/genética , Genoma Bacteriano , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Pequeño no Traducido/genética , Salmonella enterica/genética , Conjuntos de Datos como Asunto , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Escherichia coli/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Internet , ARN sin Sentido/clasificación , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Bacteriano/clasificación , ARN Bacteriano/metabolismo , ARN Mensajero/clasificación , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/clasificación , ARN Pequeño no Traducido/metabolismo , Salmonella enterica/metabolismo , Análisis de Secuencia de ARN , Programas InformáticosRESUMEN
DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes' local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.
Asunto(s)
Cromosomas Bacterianos/genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Transcripción Genética , Proteínas Bacterianas/metabolismo , Simulación por Computador , Escherichia coli/genética , Perfilación de la Expresión Génica , Genes Bacterianos , Modelos Biológicos , Regiones Promotoras Genéticas , Procesos Estocásticos , TranscriptomaRESUMEN
Motivation: Many DNA-binding proteins recognize their target sequences indirectly, by sensing DNA's response to mechanical distortion. ThreaDNA estimates this response based on high-resolution structures of the protein-DNA complex of interest. Implementing an efficient nanoscale modeling of DNA deformations involving essentially no adjustable parameters, it returns the profile of deformation energy along whole genomes, at base-pair resolution, within minutes on usual laptop/desktop computers. Our predictions can also be easily combined with estimations of direct selectivity through a generalized form of position-weight-matrices. The formalism of ThreaDNA is accessible to a wide audience. Results: We demonstrate the importance of indirect readout for the nucleosome as well as the bacterial regulators Fis and CRP. Combined with the direct contribution provided by usual sequence motifs, it significantly improves the prediction of sequence selectivity, and allows quantifying the two distinct physical mechanisms underlying it. Availability and implementation: Python software available at bioinfo.insa-lyon.fr, natively executable on Linux/MacOS systems with a user-friendly graphical interface. Galaxy webserver version available. Contact: sam.meyer@insa-lyon.fr. Supplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
Biología Computacional/métodos , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Modelos Moleculares , Programas Informáticos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Factor Proteico para Inverción de Estimulación/metabolismo , Histonas/metabolismo , Conformación de Ácido Nucleico , Nucleosomas/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/metabolismoRESUMEN
Early studies of transcriptional regulation focused on individual gene promoters defined specific transcription factors as central agents of genetic control. However, recent genome-wide data propelled a different view by linking spatially organized gene expression patterns to chromosomal dynamics. Therefore, the major problem in contemporary molecular genetics concerned with transcriptional gene regulation is to establish a unifying model that reconciles these two views. This problem, situated at the interface of polymer physics and network theory, requires development of an integrative methodology. In this review, we discuss recent achievements in classical model organism E. coli and provide some novel insights gained from studies of a bacterial plant pathogen, D. dadantii. We consider DNA topology and the basal transcription machinery as key actors of regulation, in which activation of functionally relevant genes is coupled to and coordinated with the establishment of extended chromosomal domains of coherent transcription. We argue that the spatial organization of genome plays a fundamental role in its own regulation.
Asunto(s)
Cromosomas Bacterianos , Transcripción Genética , ADN Bacteriano/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Modelos Genéticos , Operón , Plantas/microbiologíaRESUMEN
BACKGROUND: Gene expression is an inherently stochastic process, owing to its dynamic molecular nature. Protein amount distributions, which can be acquired by cytometry using a reporter gene, can inform about the mechanisms of the underlying microscopic molecular system. RESULTS: By using different clones of chicken erythroid progenitor cells harboring different integration sites of a CMV-driven mCherry protein, we investigated the dynamical behavior of such distributions. We show that, on short term, clone distributions can be quickly regenerated from small population samples with a high accuracy. On longer term, on the contrary, we show variations manifested by correlated fluctuation in the Mean Fluorescence Intensity. In search for a possible cause of this correlation, we demonstrate that in response to small temperature variations cells are able to adjust their gene expression rate: a modest (2 °C) increase in external temperature induces a significant down regulation of mean expression values, with a reverse effect observed when the temperature is decreased. Using a two-state model of gene expression we further demonstrate that temperature acts by modifying the size of transcription bursts, while the burst frequency of the investigated promoter is less systematically affected. CONCLUSIONS: For the first time, we report that transcription burst size is a key parameter for gene expression that metazoan cells from homeotherm animals can modify in response to an external thermal stimulus.
Asunto(s)
Eritroblastos/metabolismo , Células Precursoras Eritroides/metabolismo , Regulación de la Expresión Génica/fisiología , Expresión Génica/genética , Temperatura , Alpharetrovirus/genética , Animales , Línea Celular Transformada , Pollos , Citometría de Flujo , Fluorescencia , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Procesos Estocásticos , Transcripción Genética/genéticaRESUMEN
DNA torsional stress is generated by virtually all biomolecular processes involving the double helix, in particular transcription where a significant level of stress propagates over several kilobases. If another promoter is located in this range, this stress may strongly modify its opening properties, and hence facilitate or hinder its transcription. This mechanism implies that transcribed genes distant of a few kilobases are not independent, but coupled by torsional stress, an effect for which we propose the first quantitative and systematic model. In contrast to previously proposed mechanisms of transcriptional interference, the suggested coupling is not mediated by the transcription machineries, but results from the universal mechanical features of the double-helix. The model shows that the effect likely affects prokaryotes as well as eukaryotes, but with different consequences owing to their different basal levels of torsion. It also depends crucially on the relative orientation of the genes, enhancing the expression of eukaryotic divergent pairs while reducing that of prokaryotic convergent ones. To test the in vivo influence of the torsional coupling, we analyze the expression of isolated gene pairs in the Drosophila melanogaster genome. Their orientation and distance dependence is fully consistent with the model, suggesting that torsional gene coupling may constitute a widespread mechanism of (co)regulation in eukaryotes.
Asunto(s)
ADN/fisiología , Modelos Genéticos , Torsión Mecánica , Transcripción Genética/fisiología , Animales , Biología Computacional , ADN/química , ADN/genética , ADN/metabolismo , Drosophila melanogaster/genética , Levaduras/genéticaRESUMEN
Biological organisms exist over a broad temperature range of -15°C to +120°C, where many molecular processes involving DNA depend on the nanoscale properties of the double helix. Here, we present results of extensive molecular dynamics simulations of DNA oligomers at different temperatures. We show that internal basepair conformations are strongly temperature-dependent, particularly in the stretch and opening degrees of freedom whose harmonic fluctuations can be considered the initial steps of the DNA melting pathway. The basepair step elasticity contains a weaker, but detectable, entropic contribution in the roll, tilt, and rise degrees of freedom. To extend the validity of our results to the temperature interval beyond the standard melting transition relevant to extremophiles, we estimate the effects of superhelical stress on the stability of the basepair steps, as computed from the Benham model. We predict that although the average twist decreases with temperature in vitro, the stabilizing external torque in vivo results in an increase of â¼1°/bp (or a superhelical density of Δσ ≃ +0.03) in the interval 0-100°C. In the final step, we show that the experimentally observed apparent bending persistence length of torsionally unconstrained DNA can be calculated from a hybrid model that accounts for the softening of the double helix and the presence of transient denaturation bubbles. Although the latter dominate the behavior close to the melting transition, the inclusion of helix softening is important around standard physiological temperatures.
Asunto(s)
ADN/química , Elasticidad , Nanoestructuras/química , Conformación de Ácido Nucleico , Temperatura , Emparejamiento Base , Secuencia de Bases , Entropía , Timina/químicaRESUMEN
The interaction of histone H1 with linker DNA results in the formation of the nucleosomal stem structure, with considerable influence on chromatin organization. In a recent paper [Syed,S.H., Goutte-Gattat,D., Becker,N., Meyer,S., Shukla,M.S., Hayes,J.J., Everaers,R., Angelov,D., Bednar,J. and Dimitrov,S. (2010) Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome. Proc. Natl Acad. Sci. USA, 107, 9620-9625], we published results of biochemical footprinting and cryo-electron-micrographs of reconstituted mono-, di- and tri-nucleosomes, for H1 variants with different lengths of the cationic C-terminus. Here, we present a detailed account of the analysis of the experimental data and we include thermal fluctuations into our nano-scale model of the stem structure. By combining (i) crystal and NMR structures of the nucleosome core particle and H1, (ii) the known nano-scale structure and elasticity of DNA, (iii) footprinting information on the location of protected sites on the DNA backbone and (iv) cryo-electron micrographs of reconstituted tri-nucleosomes, we arrive at a description of a polymorphic, hierarchically organized stem with a typical length of 20 ± 2 base pairs. A comparison to linker conformations inferred for poly-601 fibers with different linker lengths suggests, that intra-stem interactions stabilize and facilitate the formation of dense chromatin fibers.
Asunto(s)
Modelos Moleculares , Nucleosomas/química , Fenómenos Biomecánicos , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/química , Histonas/química , Resonancia Magnética Nuclear Biomolecular , Huella de ProteínaRESUMEN
Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution *OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121-127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.
Asunto(s)
Histonas/química , Histonas/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Secuencia de Aminoácidos , Histonas/genética , Histonas/ultraestructura , Radical Hidroxilo/metabolismo , Microscopía Electrónica , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Mutación , Nucleosomas/ultraestructura , Unión ProteicaRESUMEN
In this work we analyzed protein-protein interactions (PPIs) formed by E. coli replication proteins under three disparate bacterial growth conditions. The chosen conditions corresponded to fast exponential growth, slow exponential growth and growth cessation at the stationary phase. We performed affinity purification coupled with mass spectrometry (AP-MS) of chromosomally expressed proteins (DnaA, DnaB, Hda, SeqA, DiaA, DnaG, HolD, NrdB), tagged with sequential peptide affinity (SPA) tag. Composition of protein complexes was characterized using MaxQuant software. To filter out unspecific interactions, we employed double negative control system and we proposed qualitative and quantitative data analysis strategies that can facilitate hits identification in other AP-MS datasets. Our motivation to undertake this task was still insufficient understanding of molecular mechanisms coupling DNA replication to cellular growth. Previous works suggested that such control mechanisms could involve physical interactions of replication factors with metabolic or cell envelope proteins. However, the dynamic replication protein interaction network (PIN) obtained in this study can be used to characterize links between DNA replication and various cellular processes in other contexts.