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Results for “Signal Recognition Particle”

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At least 19 recordsLinked to original sources
PubMed2000

Elongation arrest is a physiologically important function of signal recognition particle.

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.

Biological Transport↗
PubMed1983

Disassembly and reconstitution of signal recognition particle.

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL-RNA. The particle was previously shown to function in protein translocation across, and protein integration into, the endoplasmic reticulum membrane. A rapid procedure was developed to disassemble SRP into native protein and RNA components. The method utilizes unfolding of SRP with EDTA and dissociation on polycationic matrixes. SRP proteins prepared this way sediment below 7S and are inactive in activity assays. When recombined with 7SL-RNA in the presence of magnesium, the proteins are shown to reassociate stoichiometrically with 7SL-RNA to form fully active 11S SRP.

Animals↗
PubMed1983

Translocation of a lysosomal enzyme across the microsomal membrane requires signal recognition particle.

Signal recognition particle (SRP), an 11S ribonucleoprotein (Walter and Blobel (1982) Nature 299, 691-698), is required for translocation of secretory proteins across microsomal membranes (Walter and Blobel (1980) Proc. Natl. Acad. Sci. USA 77, 7112-7116) and for asymmetric integration into microsomal membranes of a transmembrane protein (Anderson et al., (1982) J. Cell Biol. 93, 501-506). We demonstrate here that SRP is also required for translocation of the lysosomal protease cathepsin D across microsomal membranes.

Animals↗
PubMed1993

GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor.

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP-dependent reaction during protein translocation is the SRP receptor-mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be < 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.

Animals↗
PubMed2003

Structure, function and evolution of the signal recognition particle.

The signal recognition particle (SRP) is a ribonucleoprotein particle essential for the targeting of signal peptide-bearing proteins to the prokaryotic plasma membrane or the eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. SRP binds to the signal peptide emerging from the exit site of the ribosome and forms a ribosome nascent chain (RNC)-SRP complex. The RNC-SRP complex then docks in a GTP-dependent manner with a membrane-anchored SRP receptor and the protein is translocated across or integrated into the membrane through a channel called the translocon. Recently considerable progress has been made in understanding the architecture and function of SRP.

Amino Acid Sequence↗
PubMed2001

Functional characterization of recombinant chloroplast signal recognition particle.

The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins. The chloroplast SRP (cpSRP) is unique among SRPs in that it possesses no RNA and is functional in post-translational as well as co-translational targeting. We have expressed and purified the two components of the Arabidopsis thaliana chloroplast signal recognition particle (cpSRP) involved in post-translational transport: cpSRP54 and the chloroplast-specific protein, cpSRP43. Recombinant cpSRP supports the efficient in vitro insertion of pea preLhcb1 into isolated thylakoid membranes. Recombinant cpSRP is a stable heterodimer with a molecular mass of approximately 100 kDa as determined by analytical ultracentrifugation, gel filtration analysis, and dynamic light scattering. The interactions of the components of the recombinant heterodimer and pea preLhcb1 were probed using an immobilized peptide library (pepscan) approach. These data confirm two previously reported interactions with the L18 region and the third transmembrane helix of Lhcb1 and suggest that the interface of the cpSRP43 and cpSRP54 proteins is involved in substrate binding. Additionally, cpSRP components are shown to recognize peptides from the cleavable, N-terminal chloroplast transit peptide of preLhcb1. The interaction of cpSRP43 with cpSRP54 was probed in a similar experiment with a peptide library representing cpSPR54. The C terminus of cpSRP54 is essential for the formation of the stable cpSRP complex and cpSPR43 interacts with distinct regions of the M domain of cpSRP54.

Amino Acid Sequence↗
PubMed1999

New insights into signal recognition and elongation arrest activities of the signal recognition particle.

The signal recognition particle (SRP), a ubiquitous cytoplasmic ribonucleoprotein particle, plays an essential role in promoting co-translational translocation of proteins into the endoplasmic reticulum. Here, we summarise recent progress made in the understanding of two essential SRP functions: the signal recognition function, which ensures the specificity, and the elongation arrest function, which increases the efficiency of translocation. Our discussion is based on functional data as well as on atomic structure information, both of which also support the notion that SRP is a very ancient particle closely related to ribosomes. Based on the significant increase of knowledge that has been accumulating on the structure of elongation factors and on their interactions with the ribosome, we speculate about a possible mechanism of the elongation arrest function.

Alu Elements↗
PubMed2002

Crystal structure of SRP19 in complex with the S domain of SRP RNA and its implication for the assembly of the signal recognition particle.

The signal recognition particle (SRP) is a ribonucleoprotein particle involved in GTP-dependent translocation of secretory proteins across membranes. In Archaea and Eukarya, SRP19 binds to 7SL RNA and promotes the incorporation of SRP54, which contains the binding sites for GTP, the signal peptide, and the membrane-bound SRP receptor. We have determined the crystal structure of Methanococcus jannaschii SRP19 bound to the S domain of human 7SL RNA at 2.9 A resolution. SRP19 clamps the tetraloops of two branched helices (helices 6 and 8) and allows them to interact side by side. Helix 6 acts as a splint for helix 8 and partially preorganizes the binding site for SRP54 in helix 8, thereby facilitating the binding of SRP54 in assembly.

Amino Acid Sequence↗
PubMed2005

RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle.

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.

Bacterial Proteins↗
PubMed2001

The cost of exposing a hydrophobic loop and implications for the functional role of 4.5 S RNA in the Escherichia coli signal recognition particle.

The signal recognition particle (SRP) is an RNA-protein complex that directs ribosomes to the rough endoplasmic reticulum membrane by binding to targeting signals found on the nascent chain of proteins destined for export to the endoplasmic reticulum. We found evidence from studies with fragments of the protein component of the Escherichia coli SRP that a long hydrophobic loop (the so-called "finger loop") is detrimental to the stability of its signal peptide-binding domain, the M domain. This hydrophobic loop is highly conserved and thus may have a critical role in the function of the SRP. Given our previously reported evidence that 4.5 S RNA stabilizes the tertiary fold of the M domain (Zheng, N., and Gierasch, L. M. (1997) Mol. Cell 1, 79-87), we now propose that the functional requirement for 4.5 S RNA resides in its ability to counteract the destabilizing influence of the finger loop.

Amino Acid Sequence↗
PubMed2000

Crystal structure of the ribonucleoprotein core of the signal recognition particle.

The signal recognition particle (SRP), a protein-RNA complex conserved in all three kingdoms of life, recognizes and transports specific proteins to cellular membranes for insertion or secretion. We describe here the 1.8 angstrom crystal structure of the universal core of the SRP, revealing protein recognition of a distorted RNA minor groove. Nucleotide analog interference mapping demonstrates the biological importance of observed interactions, and genetic results show that this core is functional in vivo. The structure explains why the conserved residues in the protein and RNA are required for SRP assembly and defines a signal sequence recognition surface composed of both protein and RNA.

Amino Acid Sequence↗
PubMed1987

Characterization of human autoantibodies that selectively precipitate the 7SL RNA component of the signal recognition particle.

The signal recognition particle (SRP), which consists of the 7SL RNA molecule associated with six polypeptides ranging between 9,000 and 72,000 m.w., mediates the translocation of newly synthesized proteins across the endoplasmic reticulum. We have characterized autoantibodies that are directed against this particle from two patients with rheumatic diseases. These sera immunoprecipitated the 7SL RNA from whole extracts of HeLa cells radiolabeled with 32P, but no RNA from deproteinized cell extracts. From 35S-methionine-labeled cell extracts, they immunoprecipitated a single polypeptide of 54,000 m.w. that is consistent with a known SRP component. Sucrose density gradient studies confirmed that this protein co-migrated with the 7SL RNA, indicating the likelihood that it is physically associated with this RNA. Thus, the 54,000 m.w. SRP protein, which is essential for the SRP functions of elongation arrest and translocation, appears to be a preferential target for human autoimmune responses. Human autoantibodies that recognize the SRP should be useful adjuncts to animal antisera for studies of the structure and function of this particle.

Arthritis↗
PubMed1987

Evidence for an extended 7SL RNA structure in the signal recognition particle.

The signal recognition particle (SRP) functions in conjunction with the SRP receptor to target nascent ectoplasmic proteins to the protein translocation machinery of the endoplasmic reticulum membrane. SRP is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of 7SL RNA 300 nucleotides long. SRP has previously been visualized by a variety of electron microscopic techniques as a rod-shaped particle 24 nm long and 6 nm wide. We report here microanalysis by electron spectroscopic imaging which localizes the RNA molecule in SRP to primarily the two ends of the particle. These results suggest that the single 7SL RNA molecule spans the length of the particle. Micrographs from a scanning transmission electron microscope permit visualization of unstained SRP with low electron exposure, as well as the direct measurement of the mol. wt of the particle. These micrographs confirm our earlier suggestion that SRP is divided into three structural domains and allow discrimination of the two ends of the structure. The results of both techniques have been combined in a model for the structure of SRP in which we propose the basic orientation of the 7SL RNA. The structure proposed is consistent with the secondary structure predicted for the RNA and with biochemical data.

Microscopy, Electron↗
PubMed2002

Towards the structure of the mammalian signal recognition particle.

The signal recognition particle (SRP) is a ubiquitous ribonucleoprotein particle involved in the co-translational targeting of proteins to membranes. Crystal structures are now available for three protein-RNA subcomplexes from the SRP, which give insights into fundamental aspects of protein-RNA recognition, the assembly of stable ribonucleoprotein particles and the mechanism of action of the SRP.

Alu Elements↗
PubMed2001

Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle.

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the endoplasmic reticulum in eukarya or to the inner membrane in prokarya. The crystal structure of the universally conserved RNA-protein core of the Escherichia coli SRP, refined here to 1.5 A resolution, revealed minor groove recognition of the 4.5 S RNA component by the M domain of the Ffh protein. Within the RNA, nucleotides comprising two phylogenetically conserved internal loops create a unique surface for protein recognition. To determine the energetic importance of conserved nucleotides for SRP assembly, we measured the affinity of the M domain for a series of RNA mutants. This analysis reveals how conserved nucleotides within the two internal loop motifs establish the architecture of the macromolecular interface and position essential functional groups for direct recognition by the protein.

Conserved Sequence↗
PubMed2001

Crystal structure of an early protein-RNA assembly complex of the signal recognition particle.

The signal recognition particle (SRP) is a universally conserved ribonucleoprotein complex that mediates the cotranslational targeting of secretory and membrane proteins to cellular membranes. A crucial early step in SRP assembly in archaea and eukarya is the binding of protein SRP19 to specific sites on SRP RNA. Here we report the 1.8 angstrom resolution crystal structure of human SRP19 in complex with its primary binding site on helix 6 of SRP RNA, which consists of a stem-loop structure closed by an unusual GGAG tetraloop. Protein-RNA interactions are mediated by the specific recognition of a widened major groove and the tetraloop without any direct protein-base contacts and include a complex network of highly ordered water molecules. A model of the assembly of the SRP core comprising SRP19, SRP54, and SRP RNA based on crystallographic and biochemical data is proposed.

Amino Acid Sequence↗
PubMed2004

Structural insights into the signal recognition particle.

The signal recognition particle (SRP) directs integral membrane and secretory proteins to the cellular protein translocation machinery during translation. The SRP is an evolutionarily conserved RNA-protein complex whose activities are regulated by GTP hydrolysis. Recent structural investigations of SRP functional domains and interactions provide new insights into the mechanisms of SRP activity in all cells, leading toward a comprehensive understanding of protein trafficking by this elegant pathway.

Animals↗
PubMed2003

S-domain assembly of the signal recognition particle.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that associates with ribosomes to mediate the targeting of membrane and secretory proteins to biological membranes. In higher eukaryotes, SRP biogenesis involves the sequential binding of SRP19 and SRP54 proteins to the S domain of 7S RNA. The recently determined crystal structures of SRP19 in complex with the S domain, and that of the ternary complex of SRP19, the S domain and the M domain of SRP54, provide insight into the molecular basis of S-domain assembly and SRP function.

Binding Sites↗