Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
2.
Int J Pharm ; 574: 118872, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31812797

RESUMO

Medical devices (indwelling) have greatly improved healthcare. Nevertheless, infections related to the use of these apparatuses continue to be a major clinical concern. Biofilms form on surfaces after bacterial adhesion, and they function as bacterial reservoirs and as resistance and tolerance factors against antibiotics and the host immune response. Technological strategies to control biofilms and bacterial adhesion, such as the use of surface coatings, are being explored more frequently, and natural peptides may promote their development. In this study, we purified and identified antibiofilm peptides from Capsicum baccatum (red pepper) using chromatography-tandem mass spectrometry, MALDI-MS, MS/MS and bioinformatics. These peptides strongly controlled biofilm formation by Staphylococcus epidermidis, the most prevalent pathogen in device-related infections, without any antibiotic activity. Furthermore, natural peptide-coated surfaces dislayed effective antiadhesive proprieties and showed no cytotoxic effects against different representative human cell lines. Finally, we determined the lead peptide predicted by Mascot and identified CSP37, which may be useful as a prime structure for the design of new antibiofilm agents. Together, these results shed light on natural Capsicum peptides as a possible antiadhesive coat to prevent medical device colonization.


Assuntos
Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Capsicum/química , Peptídeos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus epidermidis/efeitos dos fármacos , Linhagem Celular Tumoral , Materiais Revestidos Biocompatíveis/química , Células HCT116 , Humanos , Células MCF-7 , Células PC-3 , Espectrometria de Massas em Tandem/métodos
3.
J Biol Chem ; 293(18): 6637-6646, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29535188

RESUMO

Dystrophin, encoded by the DMD gene, is critical for maintaining plasma membrane integrity during muscle contraction events. Mutations in the DMD gene disrupting the reading frame prevent dystrophin production and result in severe Duchenne muscular dystrophy (DMD); in-frame internal deletions allow production of partly functional internally deleted dystrophin and result in less severe Becker muscular dystrophy (BMD). Many known BMD deletions occur in dystrophin's central domain, generally considered to be a monotonous rod-shaped domain based on the knowledge of spectrin family proteins. However, the effects caused by these deletions, ranging from asymptomatic to severe BMD, argue against the central domain serving only as a featureless scaffold. We undertook structural studies combining small-angle X-ray scattering and molecular modeling in an effort to uncover the structure of the central domain, as dystrophin has been refractory to characterization. We show that this domain appears to be a tortuous and complex filament that is profoundly disorganized by the most severe BMD deletion (loss of exons 45-47). Despite the preservation of large parts of the binding site for neuronal nitric oxide synthase (nNOS) in this deletion, computational approaches failed to recreate the association of dystrophin with nNOS. This observation is in agreement with a strong decrease of nNOS immunolocalization in muscle biopsies, a parameter related to the severity of BMD phenotypes. The structural description of the whole dystrophin central domain we present here is a first necessary step to improve the design of microdystrophin constructs toward the goal of a successful gene therapy for DMD.


Assuntos
Distrofina/química , Distrofina/genética , Deleção de Genes , Distrofia Muscular de Duchenne/genética , Sítios de Ligação , Éxons , Humanos , Simulação de Acoplamento Molecular , Distrofia Muscular de Duchenne/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Domínios Proteicos , Fases de Leitura , Espalhamento a Baixo Ângulo , Soluções , Difração de Raios X
4.
Nucleic Acids Res ; 46(6): 3211-3217, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29408956

RESUMO

During translation's elongation cycle, elongation factor G (EF-G) promotes messenger and transfer RNA translocation through the ribosome. Until now, the structures reported for EF-G-ribosome complexes have been obtained by trapping EF-G in the ribosome. These results were based on use of non-hydrolyzable guanosine 5'-triphosphate (GTP) analogs, specific inhibitors or a mutated EF-G form. Here, we present the first cryo-electron microscopy structure of EF-G bound to ribosome in the absence of an inhibitor. The structure reveals a natural conformation of EF-G·GDP in the ribosome, with a previously unseen conformation of its third domain. These data show how EF-G must affect translocation, and suggest the molecular mechanism by which fusidic acid antibiotic prevents the release of EF-G after GTP hydrolysis.


Assuntos
Proteínas de Bactérias/metabolismo , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Guanosina Trifosfato/metabolismo , Hidrólise , Modelos Moleculares , Conformação Molecular , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/ultraestrutura , Thermus thermophilus/metabolismo
5.
J Mol Biol ; 429(23): 3617-3625, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29031699

RESUMO

In bacteria, trans-translation is the main quality control mechanism for rescuing ribosomes arrested during translation. This key process is universally conserved and plays a critical role in the viability and virulence of many pathogens. We developed a reliable in vivo double-fluorescence reporter system for the simultaneous quantification of both trans-translation and the associated proteolysis activities in bacteria. The assay was validated using mutant bacteria lacking tmRNA, SmpB, and the ClpP protease. Both antisense tmRNA-binding RNA and a peptide mimicking the SmpB C-terminal tail proved to be potent inhibitors of trans-translation in vivo. The double-fluorescent reporter was also tested with KKL-35, an oxadiazole derivative that is supposed to be a promising trans-translation inhibitor, and it surprisingly turns out that trans-translation is not the only target of KKL-35 in vivo.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética
6.
Nat Struct Mol Biol ; 22(11): 914-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26479198

RESUMO

SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas/metabolismo , Proteínas de Protozoários/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Microscopia Crioeletrônica , Dictyostelium/genética , GTP Fosfo-Hidrolases/química , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Fatores de Alongamento de Peptídeos , Proteínas/química , Ribonucleoproteína Nuclear Pequena U5 , Subunidades Ribossômicas Maiores de Eucariotos/química
7.
Med Sci (Paris) ; 31(3): 282-90, 2015 Mar.
Artigo em Francês | MEDLINE | ID: mdl-25855282

RESUMO

Protein synthesis is accomplished through a process known as translation and is carried out by the ribosome, a large macromolecular complex found in every living organism. Given the huge amount of biological data that must be deciphered, it is not uncommon for ribosomes to regularly stall during the process of translation. Any disruption of this finely tuned process will jeopardize the viability of the cell. In bacteria, the main quality-control mechanism for rescuing ribosomes that undergo arrest during translation is trans-translation, which is performed by transfer-messenger RNA (tmRNA) in association with small protein B (SmPB). However, other rescue systems have been discovered recently, revealing a far more complicated network of factors dedicated to ribosome rescue. These discoveries make it possible to consider inhibition of these pathways as a very promising target for the discovery of new antibiotics.


Assuntos
Biossíntese de Proteínas , Ribossomos/fisiologia , Animais , Códon de Terminação/genética , Códon de Terminação/metabolismo , Humanos , Redes e Vias Metabólicas , Terapia de Alvo Molecular , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/uso terapêutico , Controle de Qualidade , RNA Mensageiro/metabolismo , Ribossomos/efeitos dos fármacos , Fatores de Tempo
8.
Hum Mol Genet ; 24(5): 1267-79, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25348330

RESUMO

In-frame exon deletions of the Duchenne muscular dystrophy (DMD) gene produce internally truncated proteins that typically lead to Becker muscular dystrophy (BMD), a milder allelic disorder of DMD. We hypothesized that differences in the structure of mutant dystrophin may be responsible for the clinical heterogeneity observed in Becker patients and we studied four prevalent in-frame exon deletions, i.e. Δ45-47, Δ45-48, Δ45-49 and Δ45-51. Molecular homology modelling revealed that the proteins corresponding to deletions Δ45-48 and Δ45-51 displayed a similar structure (hybrid repeat) than the wild-type dystrophin, whereas deletions Δ45-47 and Δ45-49 lead to proteins with an unrelated structure (fractional repeat). All four proteins in vitro expressed in a fragment encoding repeats 16-21 were folded in α-helices and remained highly stable. Refolding dynamics were slowed and molecular surface hydrophobicity were higher in fractional repeat containing Δ45-47 and Δ45-49 deletions compared with hybrid repeat containing Δ45-48 and Δ45-51 deletions. By retrospectively collecting data for a series of French BMD patients, we showed that the age of dilated cardiomyopathy (DCM) onset was delayed by 11 and 14 years in Δ45-48 and Δ45-49 compared with Δ45-47 patients, respectively. A clear trend toward earlier wheelchair dependency (minimum of 11 years) was also observed in Δ45-47 and Δ45-49 patients compared with Δ45-48 patients. Muscle dystrophin levels were moderately reduced in most patients without clear correlation with the deletion type. Disease progression in BMD patients appears to be dependent on the deletion itself and associated with a specific structure of dystrophin at the deletion site.


Assuntos
Distrofina/química , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Clonagem Molecular , Progressão da Doença , Éxons , Regulação da Expressão Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Pessoa de Meia-Idade , Modelos Moleculares , Distrofia Muscular de Duchenne/patologia , Estrutura Secundária de Proteína , Fases de Leitura , Estudos Retrospectivos , Deleção de Sequência , Adulto Jovem
9.
Front Microbiol ; 5: 113, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24711807

RESUMO

Ribosome stalling is a serious issue for cell survival. In bacteria, the primary rescue system is trans-translation, performed by tmRNA and its protein partner small protein B (SmpB). Since its discovery almost 20 years ago, biochemical, genetic, and structural studies have paved the way to a better understanding of how this sophisticated process takes place at the cellular and molecular levels. Here we describe the molecular details of trans-translation, with special mention of recent cryo-electron microscopy and crystal structures that have helped explain how the huge tmRNA-SmpB complex targets and delivers stalled ribosomes without interfering with canonical translation.

10.
J Mol Biol ; 426(2): 377-88, 2014 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-24095898

RESUMO

During protein synthesis, many translating ribosomes are bound together with an mRNA molecule to form polysomes (or polyribosomes). While the spatial organization of bacterial polysomes has been well studied in vitro, little is known about how they cluster when cellular conditions are highly constrained. To better understand this, we used electron tomography, template matching, and three-dimensional modeling to analyze the supramolecular network of ribosomes after induction of translational pauses. In Escherichia coli, we overexpressed an mRNA carrying a polyproline motif known to induce pausing during translation. When working with a strain lacking transfer-messenger RNA, the principle actor in the "trans-translation" rescuing system, the cells survived the hijacking of the translation machinery but this resulted in a sharp modification of the ribosomal network. The results of our experiments demonstrate that single ribosomes are replaced with large amounts of compacted polysomes. These polysomes are highly organized, principally forming hairpins and dimers of hairpins that stack together. We propose that these spatial arrangements help maintain translation efficiency when the rescue systems are absent or overwhelmed.


Assuntos
Escherichia coli/química , Escherichia coli/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Polirribossomos/química , Polirribossomos/metabolismo , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
Biochemistry ; 52(44): 7777-84, 2013 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-24063785

RESUMO

Dystrophin is a large skeletal muscle protein located at the internal face of the plasma membrane and interacting with membrane phospholipids and a number of cytosolic proteins. Binding of neuronal nitric oxide synthase (nNOS) to dystrophin appears to be crucial for exercise-induced increases in blood supply in muscle cells. By contrast, utrophin, the developmental homologous protein of dystrophin, does not display nNOS interaction. Recent in vitro and in vivo experiments showed that the dystrophin region involved in nNOS binding is located in spectrin-like repeats R16 and R17 of its filamentous central domain. Using homology modeling and atomistic molecular dynamics simulation, we compared the structural organization and surface potentials of dystrophin, utrophin, and chimeric fragments, thus revisiting the dystrophin-nNOS binding region. Our simulation results are in good agreement with experimental data. They provide a three-dimensional representation of the repeats and give insight into the molecular organization of the regions involved in dystrophin-nNOS interaction. This study also further elucidates the physical properties crucial for this interaction, particularly the presence of a large hydrophobic patch. These results will be helpful to improving our understanding of the phenotypic features of patients bearing mutations in the nNOS-binding region of dystrophin.


Assuntos
Distrofina/química , Distrofina/metabolismo , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/metabolismo , Motivos de Aminoácidos , Distrofina/genética , Humanos , Simulação de Dinâmica Molecular , Óxido Nítrico Sintase Tipo I/genética , Ligação Proteica , Estrutura Secundária de Proteína
12.
Trends Biochem Sci ; 38(8): 403-11, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23820510

RESUMO

In bacteria, the main quality control mechanism for rescuing ribosomes that have arrested during translation is trans-translation, performed by transfer-mRNA (tmRNA) associated with small protein B (SmpB). Intriguingly, this very elegant mechanism is not always necessary to maintain cell viability, suggesting the existence of alternatives. Other rescue systems have recently been discovered, revealing a far more complicated story than expected. These include the alternative ribosome rescue factors ArfA and ArfB, the elongation factors EF4 and EF-P, the peptidyl-tRNA hydrolase Pth, and several protein synthesis factors. These discoveries make it possible to describe a large network of factors dedicated to ribosome rescue, thus ensuring cell survival during stresses that induce ribosome stalling.


Assuntos
Proteínas de Bactérias/metabolismo , Modelos Biológicos , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/biossíntese , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo
13.
RNA Biol ; 10(2): 314-20, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23324601

RESUMO

A finely tuned balance of translation, storage and decay of mRNAs (mRNAs) is important for the regulation of gene expression. In eukaryotic cells, this takes place in dynamic cytoplasmic RNA-protein granules termed Processing bodies (P-bodies). In this study, by using immunoelectron tomography, 3D modeling and template matching, we analyze the size and the organization of the polysomes in the vicinity of human P-bodies. Our results show the presence of several polysomes that are compatible with a translational activity around P-bodies. Therefore, movement of mRNAs between polysomes and P-bodies can take place when the two compartments are in close contact. The presence of initiation factors in the proximity of P-bodies also suggests that translation of mRNAs can resume at the periphery of these granules.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Polirribossomos/metabolismo , Transporte de RNA , Grânulos Citoplasmáticos/genética , Tomografia com Microscopia Eletrônica , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4G em Eucariotos/genética , Fator de Iniciação 4G em Eucariotos/metabolismo , Células HeLa , Humanos , Polirribossomos/genética , Polirribossomos/ultraestrutura , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
PLoS One ; 6(8): e23819, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21901138

RESUMO

Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD). It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy.


Assuntos
Distrofina/metabolismo , Simulação de Dinâmica Molecular , Biologia Computacional , Distrofina/química , Humanos , Estrutura Terciária de Proteína
15.
Virology ; 414(1): 51-62, 2011 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-21470649

RESUMO

Despite progress in our knowledge of the internal organisation of influenza virus particles, little is known about the determinants of their morphology and, more particularly, of the actual abundance of structural proteins at the virion level. To address these issues, we used cryo-EM to focus on viral (and host) factors that might account for observed differences in virion morphology and characteristics such as size, shape and glycoprotein (GP) spike density. Twelve recombinant viruses were characterised in terms of their morphology, neuraminidase activity and virus growth. The genomic composition was shown to be important in determining the GP spike density. In particular, polymerase gene segments and especially PB1/PB2 were shown to have a prominent influence in addition to that for HA in determining GP spike density, a feature consistent with a functional link between these virus components important for virus fitness.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/ultraestrutura , Vírus da Influenza A Subtipo H3N2/ultraestrutura , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Vírion/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/ultraestrutura , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Neuraminidase/ultraestrutura , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/ultraestrutura , Vírion/metabolismo
16.
EMBO J ; 29(22): 3810-8, 2010 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-20953161

RESUMO

Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalysing peptide bond formation between amino acids. When bacterial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA-SmpB ribonucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degradation of the incomplete proteins. Here, we present the cryo-electron microscopy structures of tmRNA-SmpB accommodated or translocated into stalled ribosomes. Two atomic models for each state are proposed. This study reveals how tmRNA-SmpB crosses the ribosome and how, as the problematic mRNA is ejected, the tmRNA resume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence. This provides a structural basis for the transit of the large tmRNA-SmpB complex through the ribosome and for the means by which the tmRNA internal frame is set for translation to resume.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Thermus thermophilus/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Microscopia Crioeletrônica , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Conformação Proteica , RNA Bacteriano/química , Proteínas de Ligação a RNA/química , Ribossomos/química , Thermus thermophilus/química
17.
J Biol Chem ; 285(20): 15100-15110, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20228408

RESUMO

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg(2+)-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.


Assuntos
Biopolímeros/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Magnésio/metabolismo , Animais , Biopolímeros/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP90/química , Microscopia Eletrônica de Transmissão , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Ultracentrifugação
18.
Biol Cell ; 100(7): 413-25, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18215117

RESUMO

BACKGROUND INFORMATION: Hsp90 (90 kDa heat-shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide-binding-dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue). RESULTS AND DISCUSSION: Here, we present the first nucleotide-free structures of the entire eukaryotic Hsp90 (apo-Hsp90) obtained by small-angle X-ray scattering and single-particle cryo-EM (cryo-electron microscopy). We show that, in solution, apo-Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo-EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo-forms require large movements of the NTD (N-terminal domain) and MD (middle domain) around two flexible hinge regions. CONCLUSIONS: The present study shows, for the first time, the structure of the entire eukaryotic apo-Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.


Assuntos
Proteínas de Choque Térmico HSP90/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/ultraestrutura , Animais , Cristalografia por Raios X , Proteínas de Choque Térmico HSP90/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Soluções , Suínos
19.
Biophys J ; 89(6): 3721-40, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16169978

RESUMO

Molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide basepair steps are reported. The objective is to obtain the calculated dynamical structure for at least two copies of each case, use the results to examine issues with regard to convergence and dynamical stability of MD on DNA, and determine the significance of sequence context effects on all unique dinucleotide steps. This information is essential to understand sequence effects on DNA structure and has implications on diverse problems in the structural biology of DNA. Calculations were carried out on the 136 cases embedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs. All simulations were carried out using a well-defined state-of-the-art MD protocol, the AMBER suite of programs, and the parm94 force field. In a previous article (Beveridge et al. 2004. Biophysical Journal. 87:3799-3813), the research design, details of the simulation protocol, and informatics issues were described. Preliminary results from 15 ns MD trajectories were presented for the d(CpG) step in all 10 unique sequence contexts. The results indicated the sequence context effects to be small for this step, but revealed that MD on DNA at this length of trajectory is subject to surprisingly persistent cooperative transitions of the sugar-phosphate backbone torsion angles alpha and gamma. In this article, we report detailed analysis of the entire trajectory database and occurrence of various conformational substates and its impact on studies of context effects. The analysis reveals a possible direct correspondence between the sequence-dependent dynamical tendencies of DNA structure and the tendency to undergo transitions that "trap" them in nonstandard conformational substates. The difference in mean of the observed basepair step helicoidal parameter distribution with different flanking sequence sometimes differs by as much as one standard deviation, indicating that the extent of sequence effects could be significant. The observations reveal that the impact of a flexible dinucleotide such as CpG could extend beyond the immediate basepair neighbors. The results in general provide new insight into MD on DNA and the sequence-dependent dynamical structural characteristics of DNA.


Assuntos
Pareamento de Bases , Ilhas de CpG , DNA/química , Repetições de Microssatélites , Modelos Químicos , Modelos Moleculares , Análise de Sequência de DNA/métodos , Sequência de Bases , Simulação por Computador , DNA/análise , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Relação Estrutura-Atividade
20.
Biophys J ; 87(6): 3799-813, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15326025

RESUMO

We describe herein a computationally intensive project aimed at carrying out molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide base sequences. This initiative was undertaken by an international collaborative effort involving nine research groups, the "Ascona B-DNA Consortium" (ABC). Calculations were carried out on the 136 cases imbedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs. All MD simulations were carried out using a well-defined protocol, the AMBER suite of programs, and the parm94 force field. Phase I of the ABC project involves a total of approximately 0.6 mus of simulation for systems containing approximately 24,000 atoms. The resulting trajectories involve 600,000 coordinate sets and represent approximately 400 gigabytes of data. In this article, the research design, details of the simulation protocol, informatics issues, and the organization of the results into a web-accessible database are described. Preliminary results from 15-ns MD trajectories are presented for the d(CpG) step in its 10 unique sequence contexts, and issues of stability and convergence, the extent of quasiergodic problems, and the possibility of long-lived conformational substates are discussed.


Assuntos
DNA/química , Modelos Químicos , Modelos Moleculares , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Simulação por Computador , Cinética , Dados de Sequência Molecular , Movimento (Física) , Conformação de Ácido Nucleico , Relação Estrutura-Atividade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...