RESUMEN
Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.
Asunto(s)
Núcleo Celular , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cromosomas/genética , Genoma Fúngico , Biología Sintética/métodosRESUMEN
Polyglutamine (polyQ) diseases are rare autosomal-dominant neurodegenerative diseases associated with the expansion of glutamine-encoding triplet repeats in certain genes. To investigate the functional influence of repeat expansion on disease mechanisms, we applied a biallelic genome-engineering platform that we recently established, called Universal Knock-in System or UKiS, to develop a human cell trio, a set of three isogenic cell lines that are homozygous for two different numbers of repeats (first and second lines) or heterozygous for the two repeat numbers (third line). As an example of a polyQ disease, we chose spinocerebellar ataxia type 2 (SCA2). In a pseudodiploid human cell line, both alleles of the glutamine-encoding triplet repeat in the SCA2-causing gene, ataxin 2 or ATXN2, were first knocked in with a donor sequence encoding both thymidine kinase and either puromycin or blasticidin resistance proteins under dual drug selection. The knocked-in donor alleles were then substituted with a payload having either 22 or 76 triplet repeats in ATXN2 by ganciclovir negative selection. The two-step substitution and subsequent SNP typing and genomic sequencing confirmed that the SCA2-modeling isogenic cell trio was obtained: three clones of 22-repeat homozygotes, two clones of 22/76-repeat heterozygotes and two clones of 76-repeat homozygotes. Finally, RT-PCR and immunoblotting using the obtained clones showed that, consistent with previous observations, glutamine tract expansion reduced transcriptional and translational expression of ATXN2. The cell clones with homozygous long-repeat alleles, which are rarely obtained from patients with SCA2, showed more drastic reduction of ATXN2 expression than the heterozygous clones. This study thus demonstrates the potential of UKiS, which is a beneficial platform for the efficient development of cell models not only for polyQ diseases but also for any other genetic diseases, which may accelerate our deeper understanding of disease mechanisms and cell-based screening for therapeutic drugs.
Asunto(s)
Glutamina , Ataxias Espinocerebelosas , Humanos , Péptidos/genética , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , ProteínasRESUMEN
It seems obvious to laypeople that neurotypical humans experience color equivalently across their entire visual field. To some neuroscientists, psychologists, and philosophers, though, this claim has been met with skepticism, as neurophysiological evidence indicates the mechanisms that support color perception degrade with eccentricity. However, the argument that this entails altered color experience in peripheral vision is not universally accepted. Here, we address whether color experience is essentially equivalent between central and peripheral vision. To assess this, we will obtain similarity relationships between color experiences across the visual field using both online and laboratory-based far-field displays, while removing the confounds of saccades, memory, and expectation about color experiences. Our experiment was designed to provide clear evidence that would favor either unchanged or altered color experience relationships in the periphery. Our results are consistent with lay people's phenomenological reports: Color experiences, as probed by similarity relationships in central vision and the far field (60°), are equivalent when elicited by large stimuli. These findings challenge the widespread view in philosophy and cognitive science that peripheral color experiences are illusory, and are discussed in the context of their related neurophysiological, psychophysical, and philosophical literature.
Asunto(s)
Ilusiones , Campos Visuales , Humanos , Percepción Visual , Percepción de Color/fisiología , Movimientos Sacádicos , ColorRESUMEN
Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we establish a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of ~100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a standard platform with which to pursue the design of noncoding regions for genome writing in human cells.
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Células Madre Pluripotentes Inducidas , Retroelementos , Animales , Genómica , Humanos , Intrones/genética , Ratones , Mutagénesis/genética , Retroelementos/genética , Pez Cebra/genéticaRESUMEN
Whether or not depth perception influences brightness and/or lightness perception has been repeatedly discussed, and some studies have emphasized its importance. In addition, a small number of studies have empirically tested and shown the effect of depth inversion, such as seen in the Mach card illusion, on perceived lightness, and they interpreted such results in terms of lightness constancy. However, how perceived brightness changes contingent on depth inversion remains unexplained. Therefore, this study used the matching method to examine changes in brightness perception when depth inversion is observed. We created and used a three-dimensional (3D) concave object, composed of three sides made of card stock, which could be perceived as having two different shapes in 3D; it could be perceived as a horizontal concave object, corresponding to its actual physical structure, and as a convex standing object, similar in shape to a building. Participants observed this object as both a concave object and as a convex object, and judged the brightness of its surfaces during each observation. Our results show that the perception of the brightness of the object's surfaces clearly changed depending on the perception of depth. When the object was seen as convex, one part of the surface was perceived as darker than when the object was seen as concave, but the other part of the surface remained unchanged. Here we discuss the relationship between depth perception and brightness perception in terms of perceptual organization.
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Percepción de Profundidad , Percepción de Forma , Percepción Visual , Adulto , Femenino , Humanos , Masculino , Estimulación Luminosa , Adulto JovenRESUMEN
The principle of three-dimensional protein structure formation is a long-standing conundrum in structural biology. A globular domain of a soluble protein is formed by a network of atomic contacts among amino acid residues, but regions without intramolecular non-local contacts are often observed in the protein structure, especially in loop, linker, and peripheral segments with secondary structures. Although these regions can play key roles for protein function as interfaces for intermolecular interactions, their nature remains unclear. Here, we termed protein segments without non-local contacts as floating segments and sought them in tens of thousands of entries in the Protein Data Bank. As a result, we found that 0.72% of residues are in floating segments. Regarding secondary structural elements, coil structures are enriched in floating segments, especially for long segments. Interactions with polypeptides and polynucleotides, but not chemical compounds, are enriched in floating segments. The amino acid preferences of floating segments are similar to those of surface residues, with exceptions; the small side chain amino acids, Gly and Ala, are preferred, and some charged side chains, Arg and His, are disfavored for floating segments compared to surface residues. Our comprehensive characterization of floating segments may provide insights into understanding protein sequence-structure-function relationships.
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Bases de Datos de Proteínas , Proteínas/química , Proteínas/genética , Dominios Proteicos , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Transposons are major drivers of mammalian genome evolution. To obtain new insights into the contribution of transposons to the regulation of protein translation, we here examined how transposons affected the genesis and function of upstream open reading frames (uORFs), which serve as cis-acting elements to regulate translation from annotated ORFs (anORFs) located downstream of the uORFs in eukaryotic mRNAs. Among 39,786 human uORFs, 3,992 had ATG trinucleotides of a transposon origin, termed "transposon-derived upstream ATGs" or TuATGs. Luciferase reporter assays suggested that many TuATGs modulate translation from anORFs. Comparisons with transposon consensus sequences revealed that most TuATGs were generated by nucleotide substitutions in non-ATG trinucleotides of integrated transposons. Among these non-ATG trinucleotides, GTG and ACG were converted into TuATGs more frequently, indicating a CpG methylation-mediated process of TuATG formation. Interestingly, it is likely that this process accelerated human-specific upstream ATG formation within transposon sequences in 5' untranslated regions after divergence between human and nonhuman primates. Methylation-mediated TuATG formation seems to be ongoing in the modern human population and could alter the expression of disease-related proteins. This study shows that transposons have potentially been shaping the human proteome landscape via cis-acting uORF creation.
Asunto(s)
Metilación de ADN , Elementos Transponibles de ADN , Sistemas de Lectura Abierta , Proteoma/genética , Regiones no Traducidas 5' , Biología Computacional , Evolución Molecular , Genoma Humano , Humanos , Polimorfismo de Nucleótido Simple , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Long interspersed element 1 (L1) retrotransposon sequences are widespread in the human genome, occupying ~500,000 locations. The majority of L1s have lost their retrotransposition capability, although a significant population of human L1s maintains bidirectional transcriptional activity from the internal promoter. While the sense promoter drives transcription of the entire L1 mRNA and leads to L1 retrotransposition, the antisense promoter (ASP) transcribes L1-gene chimeric RNAs that include neighboring exon sequences. Activation mechanisms and functional impacts of L1ASP transcription are thought to vary at every L1ASP location. To explore the locus-specific regulation and function of L1ASP transcription, quantitative methodology is necessary for identifying the genomic positions of highly active L1ASPs on a genome-wide scale. Here, we employed deep-sequencing techniques and built a 3' RACE-based experimental and bioinformatics protocol, named the L1 antisense transcriptome protocol (LATRAP). In LATRAP, the PCR primer and the read mapping scheme were designed to reduce false positives and negatives, which may have been included as hits in previous cloning studies. LATRAP was here applied to the A549 human lung cancer cell line, and 313 L1ASP loci were detected to have transcriptional activity but differed in the number of mapped reads by four orders of magnitude. This indicates that transcriptional activities of the individual L1ASPs can vary greatly and that only a small population of L1ASP loci is active within individual nuclei. LATRAP is the first experimental method for ranking L1ASPs according to their transcriptional activity and will thus open a new avenue to unveiling the locus-specific biology of L1ASPs.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , Retroelementos , Transcripción Genética , Células A549 , ADN sin Sentido , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Regiones Promotoras GenéticasRESUMEN
DNA synthesis in the Bacillus subtilis cells has become possible using extra-cellular DNA. Generally, purified DNAs in a test tube have been required to introduce into the host cells for molecular cloning technology in the laboratory. We have developed a cell lysis technique for natural transformation using stable extra-cellular plasmid DNAs, in which the extra-cellular plasmid DNAs are released from lysed Escherichia coli cells. DNA synthesis then proceeds by fragment assembly using the stable extracellular DNAs, without biochemical purification. DNA synthesis of the mouse mitochondrial genome in B. subtilis genome was illustrated using four E. coli strains with plasmid DNAs carrying contiguous DNA fragments. In the natural environment, unpurified extra-cellular DNAs contribute to the gene delivery during horizontal gene transfer (HGT). The technology introduced in the present study mimics HGT and should have a wide range of applications.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , ADN/biosíntesis , Escherichia coli/metabolismo , Técnicas de Transferencia de Gen , Mitocondrias/metabolismo , Animales , ADN/química , Escherichia coli/citología , Ratones , PlásmidosRESUMEN
To perceive the external world stably, humans must integrate and manage continuous streams of information from various sensory modalities, in addition to drawing on past experiences and knowledge. In this study, we introduce a novel visuo-tactile illusion elicited by a visual-depth-reversal stimulus. The stimulus (a model of a building) was constructed so as to produce the same retinal image as an opaque cuboid, although it actually consisted of only three PVC boards forming a three-dimensional corner with the hollow inside facing the observer. Participants holding the model in their palm, therefore, observed, with both eyes or one eye, a building model that could be interpreted as either a concave or a convex cuboid. That is, tactile information from the contact surface contradicted the visual interpretation of a convex cuboid. Questionnaire and experimental results, however, showed that the building model was stably viewed as a standing cuboid, particularly under monocular observation. Participants also reported feeling a stable touch of the shrinking base of the apparently standing building model, thus ignoring the veridical contact surface. Given that the visual-tactile information was unchanged with or without the illusion and that the experimental task was tactile estimation, it is remarkable that participants failed to perceive actual touch based on the object's appearance. Results indicate the complexity and flexibility of visual-tactile integration processes. We also discuss the possibility that object knowledge influences visual-tactile integration.
Asunto(s)
Ilusiones/fisiología , Percepción del Tamaño/fisiología , Percepción del Tacto/fisiología , Percepción Visual/fisiología , Adulto , Femenino , Humanos , Masculino , Visión Binocular/fisiología , Visión Monocular/fisiología , Adulto JovenRESUMEN
The substrate specificity of cAMP-dependent protein kinase A (PKA) is controlled by its interaction with the A-kinase anchoring protein (AKAP) family. Individual AKAP members are localized to particular intracellular sites and tether PKA specifically to the subcellular compartments where target substrates exist. Here, we report that the human hypothetical gene C18orf42 encodes a novel PKA-binding protein that potentially regulates PKA-AKAP interactions. C18orf42 is expressed preferentially in neural tissues. Functional motif searching predicted that C18orf42 may encode a short protein that contains a putative PKA-binding motif. To confirm this possibility, we applied the CRISPR/Cas9 genome-editing system to incorporate the FLAG tag into the C-terminus of the endogenous C18orf42 protein in the mouse neural cell line Neuro2a. Immunoprecipitation and immunoblotting using anti-FLAG antibody showed translation of the endogenous C18orf42 protein and the physical interaction of the C18orf42 protein with PKA subunits. Immunoprecipitation and pull-down assays showed that C18orf42 binds specifically to the type II regulatory subunits of PKA. Unlike the expression of many AKAPs, that of C18orf42 could block the AKAP-mediated subcellular localization of PKA. These findings suggest that C18orf42 may be a novel PKA signaling gene that serves as an endogenous disruptor peptide for PKA-AKAP interactions.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Cromosomas Humanos Par 18 , Ingeniería Genética , Células HEK293 , Humanos , Proteínas de Transporte de Membrana , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Unión Proteica , Estructura Terciaria de Proteína/genéticaRESUMEN
L1 retrotransposons have been the major driver of structural variation of the human genome. L1 insertion polymorphism (LIP)-mediated genomic variation can alter the transcriptome and contribute to the divergence of human phenotypes. To assess this possibility, a genome-wide association study (GWAS) including LIPs is required. Toward this ultimate goal, the present study examined linkage disequilibrium between six LIPs and their neighboring single nucleotide polymorphisms (SNPs). Genomic PCR and sequencing of L1-plus and -minus alleles from different donors revealed that all six LIPs were in strong linkage disequilibrium with at least one SNP. In addition, comparison of syntenic regions containing the identified SNP nucleotides was performed among modern humans (L1-plus and -minus alleles), archaic humans and non-human primates, revealing two different evolutionary schemes that might have resulted in the observed strong SNP-LIP linkage disequilibria. This study provides an experimental framework and guidance for a future SNP-LIP integrative GWAS.
Asunto(s)
Desequilibrio de Ligamiento , Elementos de Nucleótido Esparcido Largo , Polimorfismo de Nucleótido Simple , Retroelementos , Alelos , Animales , Evolución Biológica , Genoma , Genoma Humano , Estudio de Asociación del Genoma Completo , Genotipo , Haplotipos , Heterocigoto , Hominidae , Humanos , Intrones , Nucleótidos/genética , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
Selective discrimination of a single-nucleotide difference in single-stranded DNA or RNA remains a challenge with conventional DNA or RNA probes. A peptide nucleic acid (PNA)-derived probe, in which PNA forms a pseudocomplementary heteroduplex with inosine-containing DNA or RNA, effectively discriminates a single-nucleotide difference in a closely related group of sequences of single-stranded DNA and/or RNA. The pseudocomplementary PNA heteroduplex is easily converted to a fluorescent probe that distinctively detects a member of highly homologous let-7 microRNAs.
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Colorantes Fluorescentes/síntesis química , Sondas de Ácido Nucleico/síntesis química , Ácidos Nucleicos de Péptidos/química , Polimorfismo de Nucleótido Simple , ARN/análisis , Emparejamiento Base , Secuencia de Bases , Citosina/análisis , ADN/análisis , ADN/química , ADN de Cadena Simple/análisis , Inosina/análisis , MicroARNs/análisis , Ácidos Nucleicos Heterodúplex/química , Sondas de Ácido Nucleico/química , ARN/química , ARN/genéticaRESUMEN
Adipogenesis is a well-characterized cell differentiation process. A large body of evidence has revealed the core transcription factors and signaling pathways that govern adipogenesis, but cross-talks between these cellular signals and its functional consequences have not been thoroughly investigated. We, therefore, sought to identify genes that are regulated by multiple signaling pathways during adipogenesis of human mesenchymal stem cells. Focusing on the early stage of adipogenesis, microarray analysis and quantitative RT-PCR identified 12 genes whose transcription levels were dramatically affected by the complete adipogenic induction cocktail but not by the cocktail's individual components. Expression kinetics of these genes indicate diverse mechanisms of transcriptional regulation during adipogenesis. Functional relationships between these genes and adipogenic differentiation were frequently unknown. This study thus provided novel adipogenic gene candidates that likely mediate communications among multiple signaling pathways within human mesenchymal stem cells.
Asunto(s)
Adipogénesis/genética , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Cultivadas , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de SeñalRESUMEN
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are functional RNA molecules that have recently emerged as important regulators of gene expression at the posttranscriptional or translational level. The RNA interference effects of siRNA on gene expression make it a valuable research tool for knocking down the expression of genes in mammalian cells in vitro and in vivo enabling the elucidation of molecular mechanisms underlying human diseases. Endogenous miRNAs are involved in a variety of physiological and pathological processes in humans. In this mini-review we first address the synthesis, mechanisms of action, and functions of siRNAs. Then, we focus on recent advances and technologies in miRNA and protein research of the human placenta. Next, we discuss the clinical applications of miRNA in lung cancer. We also touch on "long" noncoding RNAs from intergenic regions of the human genome. This review article is based on a presentation given at a symposium entitled Basic and Clinical Studies on Functional RNA Molecules for Advanced Medical Technologies held at Nippon Medical School in Tokyo, Japan, on November 7, 2009.
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Técnicas Genéticas , ARN/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Terapia Genética , Vectores Genéticos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , Placenta/metabolismo , Embarazo , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Proteómica , ARN/uso terapéutico , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN no Traducido/metabolismoRESUMEN
Mammalian transcriptome analysis has uncovered tens of thousands of novel transcripts of unknown function (TUFs). Classical and recent examples suggest that the majority of TUFs may underlie vital intracellular functions as non-coding RNAs because of their low coding potentials. However, only a portion of TUFs have been studied to date, and the functional significance of TUFs remains mostly uncharacterized. To increase the repertoire of functional TUFs, we screened for TUFs whose expression is controlled during differentiation of pluripotent human mesenchymal stem cells (hMSCs). The resulting six TUFs, named transcripts related to hMSC differentiation (TMDs), displayed distinct transcriptional kinetics during hMSC adipogenesis and/or osteogenesis. Structural and comparative genomic characterization suggested a wide variety of biologically active structures of these TMDs, including a long nuclear non-coding RNA, a microRNA host gene and a novel small protein gene. Moreover, the transcriptional response to established pathway activators indicated that most of these TMDs were transcriptionally regulated by each of the two key pathways for hMSC differentiation: the Wnt and protein kinase A (PKA) signaling pathways. The present study suggests that not only TMDs but also other human TUFs may in general participate in vital cellular functions with different molecular mechanisms.
Asunto(s)
Diferenciación Celular/genética , Células Madre Mesenquimatosas/metabolismo , ARN no Traducido/biosíntesis , Transducción de Señal , Adipogénesis/genética , Adulto , Secuencia de Bases , Células Cultivadas , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/citología , MicroARNs/genética , Datos de Secuencia Molecular , Osteogénesis/genética , Proteínas/genética , ARN no Traducido/genética , ARN no Traducido/fisiologíaRESUMEN
The L1 retrotransposon is the most highly successful autonomous retrotransposon in mammals. This prolific genome parasite may on occasion benefit its host through genome rearrangements or adjustments of host gene expression. In examining possible effects of L1 elements on host gene expression, we investigated whether a full-length L1 element inserted in the antisense orientation into an intron of a cellular gene may actually split the gene's transcript into two smaller transcripts: (1) a transcript containing the upstream exons and terminating in the major antisense polyadenylation site (MAPS) of the L1, and (2) a transcript derived from the L1 antisense promoter (ASP) that includes the downstream exons of the gene. Bioinformatic analysis and experimental follow-up provide evidence for this L1 "gene-breaking" hypothesis. We identified three human genes apparently "broken" by L1 elements, as well as 12 more candidate genes. Most of the inserted L1 elements in our 15 candidate genes predate the human/chimp divergence. If indeed split, the transcripts of these genes may in at least one case encode potentially interacting proteins, and in another case may encode novel proteins. Gene-breaking represents a new mechanism through which L1 elements remodel mammalian genomes.
Asunto(s)
Evolución Molecular , Regulación de la Expresión Génica , Retroelementos/genética , Animales , Elementos sin Sentido (Genética) , Secuencia de Bases , Exones , Etiquetas de Secuencia Expresada , Células HeLa , Humanos , Intrones , Datos de Secuencia Molecular , Pan troglodytes/genética , Poliadenilación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-met , Receptores de Factores de Crecimiento/genética , Transcripción GenéticaRESUMEN
Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a beta-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5'-GCCACGTGGC-3' and the native GCN4 binding 5'-ATGACGTCAT-3' sequences with almost equal affinity in the alpha-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5'-ATGAC-3' in the alpha-helical conformation, but it neither shows affinity nor helix formation with 5'-GCCAC-3'. Because native EmBP1 forms 100 times more stable complex with 5'-GCCACGTGGC-3' over 5'-ATGACGTCAT-3', our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Leucina Zippers/fisiología , Péptidos/química , Proteínas de Plantas/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Secuencia de Carbohidratos , Dicroismo Circular , Dermatoglifia del ADN/métodos , Proteínas de Unión al ADN/síntesis química , Proteínas de Unión al ADN/fisiología , Desoxirribonucleasa I/metabolismo , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/fisiología , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Group II intron RNPs are mobile genetic elements that attack and invade duplex DNA. In this work, we monitor the invasion reaction in vitro and establish a quantitative kinetic framework for the steps of this complex cascade. We find that target site specificity is achieved after DNA binding, which occurs nonspecifically. RNP searches the bound DNA before undergoing a conformational change that is associated with identification of its specific binding site. The study reveals a facile equilibrium between intron invasion and splicing, indicating that RNP invasion of top strand DNA is a relatively unfavorable event. Group II mobility must therefore depend on the trapping of invasion products, potentially through interaction of the intron-encoded protein with the DNA target and/or initiation of reverse transcription.
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ADN/metabolismo , ADN Polimerasa Dirigida por ARN , ARN/metabolismo , Retroelementos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , ADN/farmacología , Relación Dosis-Respuesta a Droga , Intrones , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Oligonucleótidos/farmacología , Factores de TiempoRESUMEN
Short peptides could potentially provide a novel element to read-out DNA sequences from the major groove. However, it is difficult to determine sequence-preference of de novo designed monomeric short peptides. Because DNS-binding affinity and specificity of short peptides are usually much lower than those of native DNA-binding proteins, determining the sequence-preference of short peptides by conventional methods utilized to deduce the target sequence of proteins often produces an unclear outcome. We report here a general strategy to defining the sequence-preference of a DNA-binding short peptide by using the heterodimers. A GCN4 basic region peptide tethers a low-affinity DNA-binding peptide adjacent to a GCN4 binding sequence through the cyclodextrin-adamantane association, thereby increasing local concentration of the low-affinity peptide on degenerated DNA sequences. An increase of the local concentration allows one to select a preferential sequence for the low-affinity DNA binding peptide. The method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of d-amino acids. The method is potentially applicable not only to artificial peptides, but also to other synthethic ligands.