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1.
Transbound Emerg Dis ; 67(6): 3061-3068, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32687668

RESUMO

Equine trypanosomosis comprises different parasitic diseases caused by protozoa of the subgenus Trypanozoon: Trypanosoma equiperdum (causative agent of dourine), Trypanosoma brucei (nagana) and Trypanosoma evansi (surra). Due to the absence of a vaccine and the lack of efficacy of the few available drugs, these diseases represent a major health and economic problem for international equine trade. Development of affordable, sensitive and specific diagnostic tests is therefore crucial to ensure the control of these diseases. Recently, it has been shown that a small RNA derived from the 7SL gene (7SL-sRNA) is produced in high concentrations in sera of cattle infected with Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei. Our objective was to determine whether 7SL-sRNA could serve as a marker of active infection in equids experimentally infected with Trypanosoma equiperdum by analysing the sensitivity, specificity and stability of the 7SL-sRNA. Using a two-step RT-qPCR, we were able to detect the presence of 7SL-sRNA between 2 and 7 days post-infection, whereas seroconversion was detected by complement fixation test between 5 and 14 days post-infection. There was a rapid loss of 7SL-sRNA signal from the blood of infected animals one day post-trypanocide treatment. The 7SL-sRNA RT-qPCR allowed an early detection of a treatment failure revealed by glucocorticoid-induced immunosuppression. In addition, the 7SL-sRNA remains detectable in positive sera after 7 days of storage at either 4°C, room temperature or 30°C, suggesting that there is no need to refrigerate serum samples before analysis. Our findings demonstrate continual detection of 7SL-sRNA over an extended period of experimental infection, with signals detected more than six weeks after inoculation. The detection of a strong and consistent 7SL-sRNA signal even during subpatent parasitemia and the early detection of treatment failure highlight the very promising nature of this new diagnostic method.


Assuntos
Mal do Coito (Veterinária)/diagnóstico , Doenças dos Cavalos/diagnóstico , RNA de Protozoário/isolamento & purificação , RNA Citoplasmático Pequeno/isolamento & purificação , Partícula de Reconhecimento de Sinal/isolamento & purificação , Trypanosoma/isolamento & purificação , Animais , Biomarcadores/análise , Testes de Fixação de Complemento/veterinária , Mal do Coito (Veterinária)/parasitologia , Feminino , França , Doenças dos Cavalos/parasitologia , Cavalos , Reação em Cadeia da Polimerase/veterinária , Tripanossomíase/diagnóstico , Tripanossomíase/parasitologia
2.
Nucleic Acids Res ; 43(18): 9017-27, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26275773

RESUMO

Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features-SRP RNA helix 8 and Srp54-in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development.


Assuntos
Cryptococcus neoformans/genética , RNA Fúngico/química , Partícula de Reconhecimento de Sinal/química , Basidiomycota/genética , Proteínas Fúngicas/química , Humanos , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , RNA Fúngico/isolamento & purificação , Partícula de Reconhecimento de Sinal/isolamento & purificação
3.
Nucleic Acids Res ; 34(8): 2374-85, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16682445

RESUMO

Alu elements are the most abundant repetitive elements in the human genome; they emerged from the signal recognition particle RNA gene and are composed of two related but distinct monomers (left and right arms). Alu RNAs transcribed from these elements are present at low levels at normal cell growth but various stress conditions increase their abundance. Alu RNAs are known to bind the cognate proteins SRP9/14. We purified synthetic Alu RNP, composed of Alu RNA in complex with SRP9/14, and investigated the effects of Alu RNPs and naked Alu RNA on protein translation. We found that the dimeric Alu RNP and the monomeric left and right Alu RNPs have a general dose-dependent inhibitory effect on protein translation. In the absence of SRP9/14, Alu RNA has a stimulatory effect on all reporter mRNAs. The unstable structure of sRight RNA suggests that the differential activities of Alu RNP and Alu RNA may be explained by conformational changes in the RNA. We demonstrate that Alu RNPs and Alu RNAs do not stably associate with ribosomes during translation and, based on the analysis of polysome profiles and synchronized translation, we show that Alu RNP and Alu RNA regulate translation at the level of initiation.


Assuntos
Elementos Alu , Iniciação Traducional da Cadeia Peptídica , RNA Citoplasmático Pequeno/fisiologia , Proteínas de Ligação a RNA/fisiologia , Partícula de Reconhecimento de Sinal/fisiologia , Sequência de Bases , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Elongação Traducional da Cadeia Peptídica , RNA Citoplasmático Pequeno/química , Proteínas de Ligação a RNA/isolamento & purificação , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/isolamento & purificação
4.
Protein Sci ; 15(6): 1290-302, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16672232

RESUMO

The signal recognition particle (SRP) plays an important role in the delivery of secretory proteins to cellular membranes. Mammalian SRP is composed of six polypeptides among which SRP68 and SRP72 form a heterodimer that has been notoriously difficult to investigate. Human SRP68 was purified from overexpressing Escherichia coli cells and was found to bind to recombinant SRP72 as well as in vitro-transcribed human SRP RNA. Polypeptide fragments covering essentially the entire SRP68 molecule were generated recombinantly or by proteolytic digestion. The RNA binding domain of SRP68 included residues from positions 52 to 252. Ninety-four amino acids near the C terminus of SRP68 mediated the binding to SRP72. The SRP68-SRP72 interaction remained stable at elevated salt concentrations and engaged approximately 150 amino acids from the N-terminal region of SRP72. This portion of SRP72 was located within a predicted tandem array of four tetratricopeptide (TPR)-like motifs suggested to form a superhelical structure with a groove to accommodate the C-terminal region of SRP68.


Assuntos
RNA/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação
5.
J Biol Chem ; 281(13): 8898-906, 2006 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-16439358

RESUMO

The eukaryotic signal recognition particle (SRP) and its receptor (SR) play a central role in co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. The SR is a heterodimeric complex assembled by the two GTPases SRalpha and SRbeta, which is membrane-anchored. Here we present the 2.45-A structure of mammalian SRbeta in its Mg2+ GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX. SRbeta is a member of the Ras-GTPase superfamily closely related to Arf and Sar1, while SRX belongs to the SNARE-like superfamily with a fold also known as longin domain. SRX binds to the P loop and the switch regions of SRbeta-GTP. The binding mode and structural similarity with other GTPase-effector complexes suggests a co-GAP (GTPase-activating protein) function for SRX. Comparison with the homologous yeast structure and other longin domains reveals a conserved adjustable hydrophobic surface within SRX which is of central importance for the SRbeta-GTP:SRX interface. A helix swap in SRX results in the formation of a dimer in the crystal structure. Based on structural conservation we present the SRbeta-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes.


Assuntos
Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Sequência Conservada , Cristalografia por Raios X , Glicina/química , Histidina/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Peptídeos/genética , Homologia de Sequência de Aminoácidos , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação
6.
J Mol Biol ; 345(4): 659-66, 2005 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-15588816

RESUMO

The signal recognition particle (SRP) is a ribonucleoprotein complex that plays a crucial role during the delivery of secretory proteins from the ribosome to the cell membrane. Among the six proteins of the eukaryotic SRP, the 72 kDa protein (SRP72) is the largest and least characterized. Polypeptides corresponding to various regions of the entire human SRP72 sequence were expressed in Escherichia coli, purified, and partially proteolyzed. Human SRP RNA bound with high affinity to a 63 amino acid residue region near the C terminus of SRP72. Mild treatment of the fragment with chymotrypsin abolished its RNA-binding activity. A conserved sequence with the consensus PDPXRWLPXXER was identified within a 56 amino acid residue RNA-binding domain. Sucrose gradient centrifugation and filter-binding analysis using mutant SRP RNAs showed that SRP72 bound to the moderately conserved portion of SRP RNA helix 5. Nine tetratricopeptide-like repeats (TPRs) poised to interact with other SRP or ribosomal proteins were predicted in the NH2-terminal region. These identifications assign two important functions to a large portion of SRP72 and demonstrate the RNA-binding capacity of the protein.


Assuntos
RNA/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Expressão Gênica , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , RNA/química , RNA/genética , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação
7.
J Bacteriol ; 185(3): 801-8, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12533455

RESUMO

The prokaryotic signal recognition particle (SRP) targeting system is a complex of two proteins, FtsY and Ffh, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic membrane cotranslationally. We previously showed that Neisseria gonorrhoeae PilA is the gonococcal FtsY homolog. In this work, we isolated the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and characterized the interactions among the three SRP components by using gel retardation and nitrocellulose filter-binding assays and enzymatic analyses of the two proteins. In the current model of prokaryotic SRP function, based on studies of the Escherichia coli and mammalian systems, Ffh binds to 4.5S RNA and the Ffh-4.5S RNA complex binds to the signal sequence of nascent peptides and then docks with FtsY at the membrane. GTP is hydrolyzed by both proteins synergistically, and the nascent peptide is transferred to the translocon. We present evidence that the in vitro properties of the gonococcal SRP differ from those of previously described systems. GTP hydrolysis by PilA, but not that by Ffh, was stimulated by 4.5S RNA, suggesting a direct interaction between PilA and 4.5S RNA that has not been reported in other systems. This interaction was confirmed by gel retardation analyses in which PilA and Ffh, both alone and together, bound to 4.5S RNA. An additional novel finding was that P(pilE) DNA, previously shown by us to bind PilA in vitro, also stimulates PilA GTP hydrolysis. On the basis of these data, we hypothesize that DNA may play a role in targeting proteins via the SRP.


Assuntos
DNA Bacteriano/fisiologia , Proteínas de Escherichia coli/fisiologia , Proteínas de Fímbrias , Guanosina Trifosfato/metabolismo , Neisseria gonorrhoeae/metabolismo , RNA Ribossômico/fisiologia , Partícula de Reconhecimento de Sinal/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência de Bases , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , GTP Fosfo-Hidrolases/metabolismo , Hidrólise , Dados de Sequência Molecular , RNA Bacteriano , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação
8.
J Bacteriol ; 184(12): 3260-7, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12029042

RESUMO

The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.


Assuntos
Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Haloferax volcanii/genética , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Proteínas Arqueais/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Haloferax volcanii/metabolismo , Dados de Sequência Molecular , RNA Arqueal/genética , RNA Arqueal/isolamento & purificação , RNA Arqueal/metabolismo , RNA Citoplasmático Pequeno/genética , RNA Citoplasmático Pequeno/isolamento & purificação , RNA Citoplasmático Pequeno/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Partícula de Reconhecimento de Sinal/isolamento & purificação
9.
FEMS Microbiol Lett ; 198(2): 105-10, 2001 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11430398

RESUMO

Protein SRP19 is an important component of the signal recognition particle (SRP) as it promotes assembly of protein SRP54 with SRP RNA and recognizes a tetranucleotide loop. Structural features and RNA binding activities of SRP19 of the hyperthermophilic archaeon Archaeoglobus fulgidus were investigated. An updated alignment of SRP19 sequences predicted three conserved regions and two alpha-helices. With Af-SRP RNA the Af-SRP54 protein assembled into an A. fulgidus SRP which remained intact for many hours. Stable complexes were formed between Af-SRP19 and truncated SRP RNAs, including a 36-residue fragment representing helix 6 of A. fulgidus SRP RNA.


Assuntos
Archaeoglobus fulgidus/genética , Conformação de Ácido Nucleico , RNA Arqueal/química , Partícula de Reconhecimento de Sinal/química , Sequência de Aminoácidos , Animais , Bactérias/genética , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Fungos/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , RNA Arqueal/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Partícula de Reconhecimento de Sinal/isolamento & purificação
10.
EMBO J ; 19(15): 4164-74, 2000 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-10921896

RESUMO

Signal recognition particle (SRP) targets proteins for co-translational insertion through or into the endoplasmic reticulum membrane. Mammalian SRP slows nascent chain elongation by the ribosome during targeting in vitro. This 'elongation arrest' activity requires the SRP9/14 subunit of the particle and interactions of the C-terminus of SRP14. We have purified SRP from Saccharomyces cerevisiae and demonstrated that it too has elongation arrest activity. A yeast SRP containing Srp14p truncated at its C-terminus (delta C29) did not maintain elongation arrest, was substantially deficient in promoting translocation and interfered with targeting by wild-type SRP. In vivo, this mutation conferred a constitutive defect in the coupling of protein translation and translocation and temperature-sensitive growth, but only a slight defect in protein translocation. In combination, these data indicate that the primary defect in SRP delta C29 is in elongation arrest, and that this is a physiologically important and conserved function of eukaryotic SRP.


Assuntos
Elongação Traducional da Cadeia Peptídica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Partícula de Reconhecimento de Sinal/metabolismo , Transporte Biológico , Compartimento Celular , Mutação , Proteínas de Ligação a RNA , Deleção de Sequência , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação
11.
Nucleic Acids Res ; 28(6): 1365-73, 2000 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-10684931

RESUMO

Signal recognition particle (SRP) takes part in protein targeting and secretion in all organisms. Searches for components of archaeal SRP in primary databases and completed genomes indicated that archaea possess only homologs of SRP RNA, and proteins SRP19 and SRP54. A recombinant SRP was assembled from cloned, expressed and purified components of the hyperthermophilic archaeon Archaeoglobus fulgidus. Recombinant Af-SRP54 associated with the signal peptide of bovine pre-prolactin translated in vitro. As in mammalian SRP, Af-SRP54 binding to Af-SRP RNA required protein Af-SRP19, although notable amounts bound in absence of Af-SRP19. Archaeoglobus fulgidus SRP proteins also bound to full-length SRP RNA of the archaeon Methanococcus jannaschii, to eukaryotic human SRP RNA, and to truncated versions which corresponded to the large domain of SRP. Dependence on SRP19 was most pronounced with components from the same species. Reconstitutions with heterologous components revealed a significant potential of human SRP proteins to bind to archaeal SRP RNAs. Surprisingly, M.jannaschii SRP RNA bound to human SRP54M quantitatively in the absence of SRP19. This is the first report of reconstitution of an archaeal SRP from recombinantly expressed purified components. The results highlight structural and functional conservation of SRP assembly between archaea and eucarya.


Assuntos
Archaeoglobus fulgidus , Proteínas de Ligação a RNA/metabolismo , RNA/genética , RNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Partícula de Reconhecimento de Sinal/metabolismo , Animais , Archaeoglobus fulgidus/química , Archaeoglobus fulgidus/genética , Azirinas , Sequência de Bases , Benzoatos , Bovinos , Clonagem Molecular , Sequência Conservada , Reagentes de Ligações Cruzadas , Humanos , Mathanococcus/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Prolactina/química , Prolactina/genética , Prolactina/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/metabolismo , RNA/biossíntese , RNA/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação
13.
Protein Sci ; 8(5): 1144-51, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10338025

RESUMO

Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; Mr = 13,584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 A, b = 63.693 A, and c = 129.601 A, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 A.


Assuntos
Metionina/química , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/isolamento & purificação , Partícula de Reconhecimento de Sinal/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Mutagênese
14.
Plant Mol Biol ; 38(3): 357-64, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9747844

RESUMO

Certain RNA molecules are known to be sequestered and stored as ribonucleoprotein particles (RNPs) in many different tissues, particularly at some stages of metabolic quiescence. In this research RNPs from embryonic axes of mature maize seeds were isolated by sucrose and CsCl gradient centrifugation and characterized based on their RNA and protein contents. Two types of RNP particles of non-ribosomal nature were identified by northern blot analysis with specific probes: the 7S RNP and the signal recognition particle (SRP) particles which contain 5S rRNA and 7S RNA respectively. The proteins associated to these RNA molecules were the transcription factor TFIIIA-homologous protein associated to 7S RNP, and the p72, p68 and p54-GTPase proteins associated to SRP.


Assuntos
Ribonucleoproteínas/metabolismo , Zea mays/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Centrifugação com Gradiente de Concentração , DNA Complementar/genética , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/isolamento & purificação , Sementes/genética , Sementes/metabolismo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação , Partícula de Reconhecimento de Sinal/metabolismo , Zea mays/embriologia , Zea mays/genética
15.
Gene ; 207(2): 197-207, 1998 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-9511762

RESUMO

Signal recognition particle (SRP) plays a critical role in the targeting of secretory proteins to cellular membranes. An essential component of SRP is the protein SRP54, which interacts not only with the nascent signal peptide, but also with the SRP RNA. To understand better how protein targeting occurs in the human system, the human SRP54 gene was cloned, sequenced, and the protein was expressed in bacteria and insect cells. Recombinant SRP54 was purified from both sources. The protein bound to SRP RNA in the presence of protein SRP19, and associated with the signal peptide of in vitro translated pre-prolactin. Comparative sequence analysis of human SRP54 with homologs from all three phylogenetic domains was combined with high-stringency protein secondary structure prediction. A conserved RNA-binding loop was predicted in the largely helical M-domain of SRP54. Contrary to general belief, the unusually high number of methionine residues clustered outside the predicted helices, thus indicating a mechanism of signal peptide recognition that may involve methionine-rich loops.


Assuntos
Partícula de Reconhecimento de Sinal/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Escherichia coli , Expressão Gênica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/isolamento & purificação , Spodoptera
16.
Nucleic Acids Res ; 25(6): 1117-22, 1997 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-9092618

RESUMO

Binding of the signal recognition particle (SRP) to signal sequences during translation leads to an inhibition of polypeptide elongation known as translation arrest. The arrest activity is mediated by a discrete domain comprised of the Alu portion of SRP RNA and a 9 and 14 kDa polypeptide heterodimer (SRP9/14). Although very few nucleotides in SRP RNA are conserved throughout evolution, the remarkable conservation of G24, which resides in the region of SRP9/14 interaction, suggests that it is essential for translation arrest. To understand the functional significance of the G24 residue, we made single base substitutions in SRP RNA at this position and analyzed the ability of the mutants to bind SRP9/14 and to reconstitute functional SRPs. Mutation of G24 to C reduced binding to SRP9/14 by at least 50-fold, whereas mutation to A and U reduced binding approximately 2- and 5-fold respectively. The mutant RNAs could nevertheless assemble into SRPs at high subunit concentrations. SRPs reconstituted with mutant RNAs were not significantly defective in translation arrest assays, indicating that the conserved guanosine does not interact directly with the translational machinery. Taken together, these results demonstrate that G24 plays an important role in the translation arrest function of SRP by mediating high affinity binding of SRP9/14.


Assuntos
Biossíntese de Proteínas , Sequências Repetitivas de Ácido Nucleico , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Primers do DNA , Dimerização , Cinética , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Mutação Puntual , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo , Partícula de Reconhecimento de Sinal/isolamento & purificação
17.
Protein Expr Purif ; 9(1): 15-26, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9116497

RESUMO

The signal recognition particle (SRP) is a ribonucleoprotein complex that mediates translocation of proteins into the endoplasmic reticulum. Protein SRP19 is an essential structural component of SRP and is believed to promote assembly of the particle. In order to have a convenient source for the purification of milligram amounts of SRP19, we expressed in Escherichia coli a human SRP19 cDNA with an amino-terminal addition of six histidine residues. Expression at 25 degrees C eliminated formation of insoluble SRP19 and resulted in accumulation of soluble hexahistidine-SRP19 to 68% of total cell protein after 24 h. Metal chelation chromatography yielded 40 mg of hexahistidine-SRP19 per liter of culture, with a purity slightly greater than 97%. To examine protein function, the RNA-binding properties of the purified protein were determined by RNA electromobility shift assays. The histidine-tagged SRP19 bound specifically to a 150-nucleotide RNA derived from SRP RNA, with an apparent Kd of 1 nM, and bound, with greatly reduced affinity, to a mutagenized form of the SRP RNA derivative that contained an altered helix 6 tetranucleotide loop. The purified protein was also photochemically crosslinked to the 150-nucleotide SRP RNA fragment, providing the means to potentially identify portions of hexahistidine-SRP19 which are in close proximity to the RNA molecule.


Assuntos
Histidina , Proteínas de Ligação a RNA/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Reagentes de Ligações Cruzadas , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Oligopeptídeos/genética , Peptídeos/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação , Raios Ultravioleta
18.
Protein Expr Purif ; 8(3): 283-94, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8936589

RESUMO

The 54-kDa subunit of the signal recognition particle (SRP) binds nascent secretory polypeptides, binds the 7SL RNA (SRP RNA) component of SRP, and hydrolyzes GTP. Limited proteolysis of SRP 54-kDa suggests the protein has two domains, termed the G (GTP-binding) and M (methionine-rich) domains. The M domain is predicted to contain a number of amphiphilic helices, which provide a binding cleft for signal sequences. In order to obtain sufficient material for studies of relationships between structure and function, we have expressed the canine cDNA encoding the 54-kDa subunit in Escherichia coli using a T7 expression system. To aid purification, the protein was expressed with an amino-terminal extension encoding an initiating methionine and 10 histidine residues followed by an enterokinase cleavage site; 0.3mg of HIS-SRP 54-kDa was purified to give a single band on SDS-PAGE in 20% yield from 500 ml of cultured E. coli. Purified HIS-SRP 54-kDa was shown to be folded into the G and M domains, to inhibit the translocation of pre-prolactin into canine microsomes, and to bind mammalian SRP RNA only in the presence of the 19-kDa subunit of SRP.


Assuntos
Proteínas Recombinantes/biossíntese , Partícula de Reconhecimento de Sinal/biossíntese , Sequência de Aminoácidos , Animais , Divisão Celular , Cães , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histidina/genética , Dados de Sequência Molecular , Plasmídeos/genética , Testes de Precipitina , Prolactina/antagonistas & inibidores , Prolactina/genética , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/isolamento & purificação
19.
FEBS Lett ; 384(3): 215-8, 1996 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-8617356

RESUMO

The SRPphi14-9 fusion protein, which can functionally replace the SRP9/14 heterodimer in the mammalian signal recognition particle (SRP), has been crystallized using the vapor diffusion method. Four different crystal forms were grown. SRPphi14-9 form IV crystals belong to the space group P4(1)22/ P4(3)22 with cell parameters a = b = 69.7 Angstroms, c = 95.7 Angstroms, alpha = beta = gamma = 90 degrees. A complete data set to 2.8 Angstroms resolution with an Rsym on intensities of 7.0% was collected on a single flash-frozen crystal.


Assuntos
Partícula de Reconhecimento de Sinal/química , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Mamíferos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Selenometionina/química , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/isolamento & purificação
20.
EMBO J ; 14(22): 5485-93, 1995 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-8521805

RESUMO

Translocation of proteins across the endoplasmic reticulum membrane is initiated by the signal recognition particle (SRP), a cytoplasmic ribonucleoprotein complex consisting of a 7S RNA and six polypeptides. To investigate the functions of the SRP components, we have tested the activities of several SRP subparticles. We show that the SRP GTPase (SRP54) alone binds a signal sequence and discriminates it from a non-signal sequence. Although SRP54 alone is unable to promote translocation, SRP54 in a complex with SRP RNA is both necessary and sufficient to promote translocation of an elongation-arrested nascent protein in a GTP-regulated manner. For co-translational translocation, additional SRP components are required. We discuss the implications of our results for the function of the Escherichia coli SRP which is homologous to the SRP54/SRP-RNA complex.


Assuntos
Sinais Direcionadores de Proteínas/metabolismo , RNA Nuclear Pequeno/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Animais , Transporte Biológico , Citosol/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Microssomos/metabolismo , Mutação , Ligação Proteica , Proteínas Recombinantes/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/isolamento & purificação
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