RESUMO
Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.
Assuntos
Transporte Ativo do Núcleo Celular , HIV-1/química , RNA Mensageiro/química , RNA Viral/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Células HEK293 , HIV-1/genética , Humanos , Dados de Sequência Molecular , Poro Nuclear/metabolismo , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
RNA conformational heterogeneity often hampers its high-resolution structure determination, especially for large and flexible RNAs devoid of stabilizing proteins or ligands. The adenosylcobalamin riboswitch exhibits heterogeneous conformations under 1 mM Mg2+ concentration and ligand binding reduces conformational flexibility. Among all conformers, we determined one apo (5.3 Å) and four holo cryo-electron microscopy structures (overall 3.0-3.5 Å, binding pocket 2.9-3.2 Å). The holo dimers exhibit global motions of helical twisting and bending around the dimer interface. A backbone comparison of the apo and holo states reveals a large structural difference in the P6 extension position. The central strand of the binding pocket, junction 6/3, changes from an 'S'- to a 'U'-shaped conformation to accommodate ligand. Furthermore, the binding pocket can partially form under 1 mM Mg2+ and fully form under 10 mM Mg2+ within the bound-like structure in the absence of ligand. Our results not only demonstrate the stabilizing ligand-induced conformational changes in and around the binding pocket but may also provide further insight into the role of the P6 extension in ligand binding and selectivity.
RESUMO
Subdomain 5BSL3.2 of hepatitis C virus RNA lies at the core of a network of distal RNA-RNA contacts that connect the 5' and 3' regions of the viral genome and regulate the translation and replication stages of the viral cycle. Using small-angle X-ray scattering and NMR spectroscopy experiments, we have determined at low resolution the structural models of this subdomain and its distal complex with domain 3'X, located at the 3'-terminus of the viral RNA chain. 5BSL3.2 adopts a characteristic 'L' shape in solution, whereas the 5BSL3.2-3'X distal complex forms a highly unusual 'Y'-shaped kissing junction that blocks the dimer linkage sequence of domain 3'X and promotes translation. The structure of this complex may impede an effective association of the viral polymerase with 5BSL3.2 and 3'X to start negative-strand RNA synthesis, contributing to explain the likely mechanism used by these sequences to regulate viral replication and translation. In addition, sequence and shape features of 5BSL3.2 are present in functional RNA motifs of flaviviruses, suggesting conserved regulatory processes within the Flaviviridae family.
Assuntos
Flaviviridae , Hepacivirus , Regiões 3' não Traduzidas , Genoma Viral , Hepacivirus/genética , Modelos Estruturais , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Replicação Viral/genéticaRESUMO
Quorum sensing gene expression in vibrios is regulated by the LuxR/HapR family of transcriptional factors, which includes Vibrio vulnificus SmcR. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic but highly degenerate with sequence variations at each promoter. To examine the mechanism by which SmcR recognizes diverse DNA sites, we generated SmcR separation-of-function mutants that either repress or activate transcription but not both. SmcR N55I is restricted in recognition of single base-pair variations in DNA binding site sequences and thus is defective at transcription activation but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R and N142D substitutions disrupt the interaction with RNAP alpha but retain functional DNA binding activity. X-ray crystallography and small angle X-ray scattering data show that the SmcR DNA binding domain exists in two conformations (wide and narrow), and the protein complex forms a mixture of dimers and tetramers in solution. The three RNAP interaction-deficient variants also have two DNA binding domain conformations, whereas SmcR N55I exhibits only the wide conformation. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNAP and a multi-conformational DNA binding domain that permits recognition of variable DNA sites.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Transativadores/química , Transativadores/genética , Fatores de Transcrição/química , Vibrio vulnificus/química , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Dimerização , Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Modelos Moleculares , Mutação , Regiões Promotoras Genéticas , Conformação Proteica , Percepção de Quorum/genética , Proteínas Recombinantes , Proteínas Repressoras/química , Proteínas Repressoras/genética , Espalhamento a Baixo Ângulo , Fatores de Transcrição/genética , Vibrio vulnificus/genéticaRESUMO
Effective gene regulation by the tetrahydrofolate riboswitch depends not only on ligand affinity but also on the kinetics of ligand association, which involves two cooperative binding sites. We have determined a 1.9-Å resolution crystal structure of the ligand-free THF riboswitch aptamer. The pseudoknot binding site 'unwinds' in the absence of ligand, whereby the adjacent helical domains (P1, P2, and P3) become disjointed, resulting in rotation and misalignment of the gene-regulatory P1 helix with respect to P3. In contrast, the second binding site at the three-way junction, which is the first to fold, is structurally conserved between apo and holo forms. This suggests a kinetic role for this site, in which binding of the first ligand molecule to the stably folded three-way junction promotes formation of the regulatory pseudoknot site and subsequent binding of the second molecule. As such, these findings provide a molecular basis for both conformational switching and kinetic control.
Assuntos
Riboswitch/genética , Tetra-Hidrofolatos/genética , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação/genética , Cristalografia por Raios X/métodos , Cinética , Ligantes , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch for nuclear magnetic resonance spectroscopy or single-molecule Förster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection, and disease diagnostics.
Assuntos
Fluorescência , Marcação por Isótopo/métodos , RNA/química , RNA/síntese química , Adenina/análise , Adenina/química , Adenina/metabolismo , Aptâmeros de Nucleotídeos/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Automação/métodos , Sequência de Bases , Técnicas Biossensoriais , DNA/genética , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/análise , RNA/genética , Riboswitch/genética , Robótica , Moldes Genéticos , Transcrição GênicaRESUMO
Histone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. The catalytic activity of this family is dependent on interactions with additional conserved proteins, but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering, cross-linking mass spectrometry, nuclear magnetic resonance spectroscopy and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 ß-propeller domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.
Assuntos
Proteínas de Ligação a DNA/química , Histona-Lisina N-Metiltransferase/química , Histonas/química , Proteína de Leucina Linfoide-Mieloide/química , Catálise , Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisina/genética , Metilação , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteína de Leucina Linfoide-Mieloide/genética , Domínios PR-SET/genética , Conformação Proteica , Mapas de Interação de Proteínas/genética , Repetições WD40/genéticaRESUMO
Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.
Assuntos
Transplante de Coração , Hidrogéis , Animais , Humanos , Imunomodulação , Camundongos , PeptídeosRESUMO
BACKGROUND This study analyzed the epidemiological characteristics and trends of trauma injuries in Ruijin Hospital North, Shanghai, China, and the feasibility of methods to prevent trauma. MATERIAL AND METHODS In this retrospective cross-sectional study, the electronic databases of Ruijin Hospital North were searched for patients who experienced severe trauma from 2013 to 2016. Characteristics of severe trauma were analyzed, including trauma mechanism, gender, reasons for injury, and injury-associated causes of death. RESULTS Of the 17,093 patients who experienced trauma during the study period, 11,165 (65.3%) were male and 5,928 (34.7%) were female. Analysis by age showed that the highest incidence of traumatic injuries was in subjects aged 25-34 years, whereas analysis by occupation showed the highest incidence of injury in migrant workers without higher education. Classification by Injury Severity Score (ISS) showed that 12,563 (73.5%) subjects had minor injuries, 4,273 (25.0%) had serious injuries, and 256 (1.5%) had severe injuries. In addition, 256 (1.5%) subjects died, with traffic accidents and falling injuries being the main causes of death. The incidence of injury peaked at 9-11 am and 2-4 pm and was significantly higher in autumn and winter than in spring and summer. CONCLUSIONS Most trauma patients were young adults. Injuries due to traffic accidents and falling were the main causes of death, with disregard of driving regulations and other health and safety regulations being the main cause of trauma. Trauma injuries may be prevented by strengthening education and by obeying traffic laws and construction site safety regulations.
Assuntos
Serviço Hospitalar de Emergência/estatística & dados numéricos , Serviço Hospitalar de Emergência/tendências , Ferimentos e Lesões/epidemiologia , Ferimentos e Lesões/etiologia , Adolescente , Adulto , Idoso , China/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s-1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s-1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s-1. The µs-ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.
Assuntos
Adenina/química , Aptâmeros de Nucleotídeos/química , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Riboswitch , Cristalografia por Raios X , Modelos MolecularesRESUMO
The 3'X domain is a 98-nt region located at the 3' end of hepatitis C virus genomic RNA that plays essential functions in the viral life cycle. It contains an absolutely conserved, 16-base palindromic sequence that promotes viral RNA dimerization, overlapped with a 7-nt tract implicated in a distal contact with a nearby functional sequence. Using small angle X-ray scattering measurements combined with model building guided by NMR spectroscopy, we have studied the stoichiometry, structure, and flexibility of domain 3'X and two smaller subdomain sequences as a function of ionic strength, and obtained a three-dimensional view of the full-length domain in its monomeric and dimeric states. In the monomeric form, the 3'X domain adopted an elongated conformation containing two SL1' and SL2' double-helical stems stabilized by coaxial stacking. This structure was significantly less flexible than that of isolated subdomain SL2' monomers. At higher ionic strength, the 3'X scattering envelope nearly doubled its size, reflecting the formation of extended homodimers containing an antiparallel SL2' duplex flanked by coaxially stacked SL1' helices. Formation of these dimers could initialize and/or regulate the packaging of viral RNA genomes into virions.
Assuntos
Regiões 3' não Traduzidas , Hepacivirus/genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Dimerização , Sequências Repetidas Invertidas , Espectroscopia de Ressonância Magnética , Concentração Osmolar , SoluçõesRESUMO
HIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occurs is not known. The Rev assembly domain has a helical-hairpin structure which associates through three (A-A, B-B and C-C) interfaces. The RRE has the topology of an upper-case letter A, with the two known Rev binding sites mapping onto the legs of the A. We have determined a crystal structure for the Rev assembly domain at 2.25â¯Å resolution, without resort to either mutations or chaperones. It shows that B-B dimers adopt an arrangement reversed relative to that previously reported, and join through a C-C interface to form tetramers. The new subunit arrangement shows how four Rev molecules can assemble on the two sites on the RRE to form the specificity checkpoint, and how further copies add through A-A interactions. Residues at the C-C interface, specifically the Pro31-Trp45 axis, are a potential target for intervention.
Assuntos
Genes env/fisiologia , HIV-1/genética , HIV-1/metabolismo , RNA Viral/metabolismo , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Genes env/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Splicing de RNA/genética , Splicing de RNA/fisiologia , RNA Viral/genéticaRESUMO
UHRF1 is a key mediator of inheritance of epigenetic DNA methylation patterns during cell division and is a putative target for cancer therapy. Recent studies indicate that interdomain interactions critically influence UHRF1's chromatin-binding properties, including allosteric regulation of its histone binding. Here, using an integrative approach that combines small angle X-ray scattering, NMR spectroscopy, and molecular dynamics simulations, we characterized the dynamics of the tandem tudor domain-plant homeodomain (TTD-PHD) histone reader module, including its 20-residue interdomain linker. We found that the apo TTD-PHD module in solution comprises a dynamic ensemble of conformers, approximately half of which are compact conformations, with the linker lying in the TTD peptide-binding groove. These compact conformations are amenable to cooperative, high-affinity histone binding. In the remaining conformations, the linker position was in flux, and the reader adopted both extended and compact states. Using a small-molecule fragment screening approach, we identified a compound, 4-benzylpiperidine-1-carboximidamide, that binds to the TTD groove, competes with linker binding, and promotes open TTD-PHD conformations that are less efficient at H3K9me3 binding. Our work reveals a mechanism by which the dynamic TTD-PHD module can be allosterically targeted with small molecules to modulate its histone reader function for therapeutic or experimental purposes.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Epigênese Genética , Histonas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Ubiquitina-Proteína Ligases , Difração de Raios XRESUMO
The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using small-angle X-ray scattering (SAXS) and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other.IMPORTANCE Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev response element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is "A" shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function.
RESUMO
Our knowledge about the functional diversity and importance of RNA in biology has grown enormously over the past three decades and has driven efforts to develop better tools to characterize RNAs. Amongst these tools are methods for preparing specifically labeled or chemically modified RNAs, which are essential for basic research, biomedical, and clinical applications. Understanding the potential and limits of these different RNA synthesis and labeling strategies is important in deciding how to approach the preparation of a particular RNA molecule. Here, we review these various labeling methods and future directions of the field.
Assuntos
RNA/genética , Coloração e Rotulagem/métodos , HumanosRESUMO
In Gram-positive bacteria, the tRNA-dependent T-box riboswitch system regulates expression of amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. Binding of uncharged tRNA "closes" the switch, allowing transcription read-through. Structural studies of the 100-nucleotide stem I domain reveal tRNA utilizes base pairing and stacking interactions to bind the stem, but little is known structurally about the 180-nucleotide riboswitch core (stem I, stem III, and antiterminator stem) in complex with tRNA or the mechanism of coupling of the intermolecular binding domains crucial to T-box function. Here we utilize solution structural and biophysical methods to characterize the interplay of the different riboswitch-tRNA contact points using Bacillus subtilis and Oceanobacillus iheyensis glycyl T-box and T-box:tRNA constructs. The data reveal that tRNA:riboswitch core binding at equilibrium involves only Specifier-anticodon and antiterminator-acceptor stem pairing. The elbow:platform stacking interaction observed in studies of the T-box stem I domain is released after pairing between the acceptor stem and the bulge in the antiterminator helix. The results are consistent with the model of T-box riboswitch:tRNA function in which tRNA is captured by stem I of the nascent mRNA followed by stabilization of the antiterminator helix and the paused transcription complex.
Assuntos
Bacillaceae/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Riboswitch , Bacillaceae/química , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA de Transferência/química , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
UNLABELLED: HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE: Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation.
Assuntos
HIV-1/fisiologia , Multimerização Proteica , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Humanos , Estrutura Secundária de ProteínaRESUMO
Incorporation of modified or labeled nucleotides at specific sites in RNAs is critical for gaining insights into the structure and function of RNAs. Preparation of site-specifically labeled large RNAs in amounts suitable for structural or functional studies is extremely difficult using current methodologies. The position-selective labeling of RNA, PLOR, is a recently developed method that makes such syntheses possible. PLOR allows incorporation of various probes, including (2)D/(13)C/(15)N-isotopic labels, Cy3/Cy5/Alexa488/Alexa555 fluorescent dyes, biotin and other chemical groups, into specific positions in long RNAs. Here, we describe in detail the use of PLOR to label RNAs at specific segment(s) or discrete sites.
Assuntos
RNA/química , Sequência de Bases , Biocatálise , RNA Polimerases Dirigidas por DNA/química , Corantes Fluorescentes/química , Hidrazinas/química , Sequências Repetidas Invertidas , Coloração e Rotulagem , Proteínas Virais/químicaRESUMO
Detailed understanding of the structure and function relationship of RNA requires knowledge about RNA three-dimensional (3D) topological folding. However, there are very few unique RNA entries in structure databases. This is due to challenges in determining 3D structures of RNA using conventional methods, such as X-ray crystallography and NMR spectroscopy, despite significant advances in both of these technologies. Computational methods have come a long way in accurately predicting the 3D structures of small (<50nt) RNAs to within a few angstroms compared to their native folds. However, lack of an apparent correlation between an RNA primary sequence and its 3D fold ultimately limits the success of purely computational approaches. In this context, small angle X-ray scattering (SAXS) serves as a valuable tool by providing global shape information of RNA. In this article, we review the progress in determining RNA 3D topological structures, including a new method that combines secondary structural information and SAXS data to sample conformations generated through hierarchical moves of commonly observed RNA motifs.
Assuntos
Modelos Moleculares , RNA/química , Sequência de Bases , Simulação por Computador , Conformação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Protein phosphorylation mediates essentially all aspects of cellular life. In humans, this is achieved by â¼500 kinases, each recognizing a specific consensus motif (CM) in the substrates. The majority of CMs are surface-exposed and are thought to be accessible to kinases for phosphorylation. Here we investigated the archetypical protein kinase A (PKA)-mediated phosphorylation of filamin, a major cytoskeletal protein that can adopt an autoinhibited conformation. Surprisingly, autoinhibited filamin is refractory to phosphorylation by PKA on a known Ser(2152) site despite its CM being exposed and the corresponding isolated peptide being readily phosphorylated. Structural analysis revealed that although the CM fits into the PKA active site its surrounding regions sterically clash with the kinase. However, upon ligand binding, filamin undergoes a conformational adjustment, allowing rapid phosphorylation on Ser(2152). These data uncover a novel ligand-induced conformational switch to trigger filamin phosphorylation. They further suggest a substrate shape-dependent filtering mechanism that channels specific exposed CM/kinase recognition in diverse signaling responses.