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1.
Cell Rep ; 36(2): 109350, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260909

RESUMO

Co-translational protein targeting to membranes by the signal recognition particle (SRP) is a universally conserved pathway from bacteria to humans. In mammals, SRP and its receptor (SR) have many additional RNA features and protein components compared to the bacterial system, which were recently shown to play regulatory roles. Due to its complexity, the mammalian SRP targeting process is mechanistically not well understood. In particular, it is not clear how SRP recognizes translating ribosomes with exposed signal sequences and how the GTPase activity of SRP and SR is regulated. Here, we present electron cryo-microscopy structures of SRP and SRP·SR in complex with the translating ribosome. The structures reveal the specific molecular interactions between SRP and the emerging signal sequence and the elements that regulate GTPase activity of SRP·SR. Our results suggest the molecular mechanism of how eukaryote-specific elements regulate the early and late stages of SRP-dependent protein targeting.


Assuntos
Mamíferos/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Animais , Bactérias/metabolismo , Microscopia Crioeletrônica , GTP Fosfo-Hidrolases/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Domínios Proteicos , Transporte Proteico , RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/ultraestrutura , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/ultraestrutura , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/ultraestrutura
2.
J Mol Biol ; 432(24): 166708, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33188783

RESUMO

The 43 kDa subunit of the chloroplast signal recognition particle, cpSRP43, is an ATP-independent chaperone essential for the biogenesis of the light harvesting chlorophyll-binding proteins (LHCP), the most abundant membrane protein family on earth. cpSRP43 is activated by a stromal factor, cpSRP54, to more effectively capture and solubilize LHCPs. The molecular mechanism underlying this chaperone activation is unclear. Here, a combination of hydrogen-deuterium exchange, electron paramagnetic resonance, and NMR spectroscopy experiments reveal that a disorder-to-order transition of the ankyrin repeat motifs in the substrate binding domain of cpSRP43 drives its activation. An analogous coil-to-helix transition in the bridging helix, which connects the ankyrin repeat motifs to the cpSRP54 binding site in the second chromodomain, mediates long-range allosteric communication of cpSRP43 with its activating binding partner. Our results provide a molecular model to explain how the conformational dynamics of cpSRP43 enables regulation of its chaperone activity and suggest a general mechanism by which ATP-independent chaperones with cooperatively folding domains can be regulated.


Assuntos
Trifosfato de Adenosina/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Membrana/genética , Partícula de Reconhecimento de Sinal/genética , Sequência de Aminoácidos/genética , Proteínas de Arabidopsis/ultraestrutura , Sítios de Ligação , Cloroplastos/genética , Complexos de Proteínas Captadores de Luz/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Ligação Proteica/genética , Conformação Proteica , Dobramento de Proteína , Partícula de Reconhecimento de Sinal/ultraestrutura
3.
Biol Chem ; 401(1): 63-80, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31408431

RESUMO

Co-translational protein targeting to membranes relies on the signal recognition particle (SRP) system consisting of a cytosolic ribonucleoprotein complex and its membrane-associated receptor. SRP recognizes N-terminal cleavable signals or signal anchor sequences, retards translation, and delivers ribosome-nascent chain complexes (RNCs) to vacant translocation channels in the target membrane. While our mechanistic understanding is well advanced for the small bacterial systems it lags behind for the large bacterial, archaeal and eukaryotic SRP variants including an Alu and an S domain. Here we describe recent advances on structural and functional insights in domain architecture, particle dynamics and interplay with RNCs and translocon and GTP-dependent regulation of co-translational protein targeting stimulated by SRP RNA.


Assuntos
Membrana Celular/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Partícula de Reconhecimento de Sinal/genética , Elementos Alu/genética , Archaea/genética , Bactérias/genética , Membrana Celular/ultraestrutura , Células Eucarióticas/metabolismo , Proteínas de Membrana/ultraestrutura , Domínios Proteicos/genética , Modificação Traducional de Proteínas/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Partícula de Reconhecimento de Sinal/ultraestrutura
4.
Curr Microbiol ; 74(2): 284-297, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27900448

RESUMO

The signal recognition particle (SRP) and its receptor constitute universally conserved and essential cellular machinery that controls the proper membrane localization of nascent polypeptides with the transmembrane domain. In the past decade, there has been an immense advancement in our understanding of this targeting machine in all three domains of life. A significant portion of such progress came from the structural analysis of archaeal SRP components. Despite the availability of structural insights from different archaeal SRP components, little is known about protein translocation in this domain of life compared to either bacteria or eukaryotes. One of the primary reasons being limited availability of the genetic and cell biological tools in archaea. In the present review, an attempt has been made to explore the structural information available for archaeal SRP components to gain insights into the protein translocation mechanism of this group of organisms. Besides, many exciting avenues of archaeal research possible using the recently developed genetic and cell biological tools for some species have been identified.


Assuntos
Archaea/fisiologia , Proteínas Arqueais/metabolismo , Proteínas de Membrana/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Archaea/metabolismo , Archaea/ultraestrutura , Transporte Proteico , Partícula de Reconhecimento de Sinal/ultraestrutura
5.
RNA ; 20(12): 1955-62, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25336584

RESUMO

The signal recognition particle (SRP) is a conserved ribonucleoprotein particle that targets membrane and secreted proteins to translocation channels in membranes. In eukaryotes, the Alu domain, which comprises the 5' and 3' extremities of the SRP RNA bound to the SRP9/14 heterodimer, is thought to interact with the ribosome to pause translation elongation during membrane docking. We present the 3.2 Å resolution crystal structure of a chimeric Alu domain, comprising Alu RNA from the archaeon Pyrococcus horikoshii bound to the human Alu binding proteins SRP9/14. The structure reveals how intricate tertiary interactions stabilize the RNA 5' domain structure and how an extra, archaeal-specific, terminal stem helps constrain the Alu RNA into the active closed conformation. In this conformation, highly conserved noncanonical base pairs allow unusually tight side-by-side packing of 5' and 3' RNA stems within the SRP9/14 RNA binding surface. The biological relevance of this structure is confirmed by showing that a reconstituted full-length chimeric archaeal-human SRP is competent to elicit elongation arrest in vitro. The structure will be useful in refining our understanding of how the SRP Alu domain interacts with the ribosome.


Assuntos
Conformação de Ácido Nucleico , Partícula de Reconhecimento de Sinal/química , Cristalografia por Raios X , Humanos , Conformação Molecular , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas/genética , Estrutura Terciária de Proteína/genética , Pyrococcus horikoshii/genética , Dobramento de RNA/genética , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/ultraestrutura
7.
Mol Biol Cell ; 17(12): 5063-74, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16987964

RESUMO

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.


Assuntos
Escherichia coli/ultraestrutura , Microscopia Eletrônica de Transmissão e Varredura/métodos , Partícula de Reconhecimento de Sinal/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Microscopia Eletrônica de Transmissão por Filtração de Energia , Modelos Moleculares , Estrutura Terciária de Proteína , Subunidades Proteicas/química , RNA Bacteriano/química , Ribossomos/metabolismo , Soluções
8.
Proc Natl Acad Sci U S A ; 101(21): 7902-6, 2004 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-15148364

RESUMO

Trigger factor (TF) and signal recognition particle (SRP) bind to the bacterial ribosome and are both crosslinked to protein L23 at the peptide exit, where they interact with emerging nascent peptide chains. It is unclear whether TF and SRP exclude one another from their ribosomal binding site(s). Here we show that SRP and TF can bind simultaneously to ribosomes or ribosome nascent-chain complexes exposing a SRP-specific signal sequence. Based on changes of the crosslinking pattern and on results obtained by fluorescence measurements using fluorescence-labeled SRP, TF binding induces structural changes in the ribosome-SRP complex. Furthermore, we show that binding of the SRP receptor, FtsY, to ribosome-bound SRP excludes TF from the ribosome. These results suggest that TF and SRP sample nascent chains on the ribosome in a nonexclusive fashion. The decision for ribosome nascent-chain complexes exposing a signal sequence to enter SRP-dependent membrane targeting seems to be determined by the binding of SRP, which is stabilized by signal sequence recognition, and promoted by the exclusion of TF due to the binding of the SRP receptor to ribosome-bound SRP.


Assuntos
Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/metabolismo , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Reagentes de Ligações Cruzadas , Escherichia coli , Proteínas de Escherichia coli/ultraestrutura , Fluorescência , Substâncias Macromoleculares , Modelos Biológicos , Peptidilprolil Isomerase/ultraestrutura , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/ultraestrutura , Ribossomos/química , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/ultraestrutura
9.
Nature ; 427(6977): 808-14, 2004 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-14985753

RESUMO

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. Here we present the structure of a targeting complex consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. This structure, solved to 12 A by cryo-electron microscopy, enables us to generate a molecular model of SRP in its functional conformation. The model shows how the S domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu domain reaches into the elongation-factor-binding site of the ribosome, explaining its elongation arrest activity.


Assuntos
Elongação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Microscopia Crioeletrônica , Mamíferos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ribossomos/química , Ribossomos/ultraestrutura , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/ultraestrutura , Relação Estrutura-Atividade
10.
Plant J ; 29(5): 531-43, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11874567

RESUMO

Biochemical and genetic studies have established that the light-harvesting chlorophyll proteins (LHCPs) of the photosystems use the cpSRP (chloroplast signal recognition particle) pathway for their targeting to thylakoids. Previous analyses of single cpSRP mutants, chaos and ffc, deficient in cpSRP43 and cpSRP54, respectively, have revealed that half of the LHCPs are still integrated into the thylakoid membranes. Surprisingly, the effects of both mutations are additive in the double mutant ffc/chaos described here. This mutant has pale yellow leaves at all stages of growth and drastically reduced levels of all the LHCPs except Lhcb 4. Although the chloroplasts have a normal shape, the thylakoid structure is affected by the mutation, probably as a consequence of reduction of all the LHCPs. ELIPs (early light-inducible proteins), nuclear-encoded proteins related to the LHCP family and inducible by light stress, were also drastically reduced in the double mutant. However, proteins targeted by other chloroplastic targeting pathways (DeltapH, Sec and spontaneous pathways) accumulated to similar levels in the wild-type and the double mutant. Therefore, the near total loss of LHCPs and ELIPs in the double mutant suggests that cpSRP is the predominant, if not exclusive, targeting pathway for these proteins. Phenotypic analysis of the double mutant, compared to the single mutants, suggests that the cpSRP subunits cpSRP43 and cpSRP54 contribute to antenna targeting in an independent but additive way.


Assuntos
Proteínas de Arabidopsis , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Proteínas de Plantas/genética , Proteínas de Saccharomyces cerevisiae , Partícula de Reconhecimento de Sinal/genética , Tilacoides/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Clorofila/efeitos da radiação , Proteínas de Cloroplastos , Complexos de Proteínas Captadores de Luz , Microscopia Eletrônica , Mutação , Fenótipo , Proteínas de Plantas/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Partícula de Reconhecimento de Sinal/ultraestrutura , Tilacoides/ultraestrutura
11.
J Struct Biol ; 113(1): 35-46, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7880651

RESUMO

The 54-kDa subunit SRP54 of the signal recognition particle in eukaryotic cells is responsible for the recognition of nascent proteins destined for secretion or membrane integration. The three-dimensional structure of this protein was determined using computational techniques applied to images of the molecule obtained via high-resolution, low-dose, scanning transmission electron microscopy at low temperature. The reconstructions at spatial resolutions between 12 and 15 A feature two unequal domains joined by a slender linker. The two-domain structure is in agreement with genetic and biochemical data indicating organization of SRP54 into a larger N-terminal GTP-binding region and a smaller C-terminal peptide-binding region. The structure has similarities to other protein domains with related functions and similar amino acid sequences. The larger domain of the 3D reconstruction is consistent in shape and size with the GTP-binding domains of EF-Tu and p21-RAS, while the smaller domain is compatible in structure with part of the peptide-binding protein calmodulin. The overall shape of SRP54 and the deduced location of critical functional regions of the molecule provide a structural framework for its known biochemical properties in the targeting cycle of the signal recognition particle.


Assuntos
Conformação Proteica , Estrutura Secundária de Proteína , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/ultraestrutura , Sequência de Aminoácidos , Calmodulina/química , Simulação por Computador , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/ultraestrutura , Microscopia Eletrônica de Transmissão e Varredura/métodos , Modelos Moleculares , Dados de Sequência Molecular , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/ultraestrutura , Relação Estrutura-Atividade
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