SEARCH · PubMed Health

Results for “Signal Recognition Particle”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources

PubMed2000

Role of SRP19 in assembly of the Archaeoglobus fulgidus signal recognition particle.

Previous studies have shown that SRP19 promotes association of the highly conserved signal peptide-binding protein, SRP54, with the signal recognition particle (SRP) RNA in both archaeal and eukaryotic model systems. In vitro characterization of this process is now reported using recombinantly expressed components of SRP from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidis. A combination of native gel mobility shift, filter binding, and Ni-NTA agarose bead binding assays were used to determine the binding constants for binary and ternary complexes of SRP proteins and SRP RNA. Archaeal SRP54, unlike eukaryotic homologues, has significant intrinsic affinity for 7S RNA (K(D) approximately 15 nM), making it possible to directly compare particles formed in the presence and absence of SRP19 and thereby assess the precise role of SRP19 in the assembly process. Chemical modification studies using hydroxyl radicals and DEPC identify nonoverlapping primary binding sites for SRP19 and SRP54 corresponding to the tips of helix 6 and helix 8 (SRP19) and the distal loop and asymmetric bulge of helix 8 (SRP54). SRP19 additionally induces conformational changes concentrated in the proximal asymmetric bulge of helix 8. Selected nucleotides in this bulge become modified as a result of SRP19 binding but are subsequently protected from modification by formation of the complete complex with SRP54. Together these results suggest a model for assembly in which bridging the ends of helix 6 and helix 8 by SRP19 induces a long-range structural change to present the proximal bulge in a conformation compatible with high-affinity SRP54 binding.

Archaeal Proteins↗

PubMed2004

Trigger factor binds to ribosome-signal-recognition particle (SRP) complexes and is excluded by binding of the SRP receptor.

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.

Bacterial Proteins↗

PubMed2002

Getting on target: the archaeal signal recognition particle.

Protein translocation begins with the efficient targeting of secreted and membrane proteins to complexes embedded within the membrane. In Eukarya and Bacteria, this is achieved through the interaction of the signal recognition particle (SRP) with the nascent polypeptide chain. In Archaea, homologs of eukaryal and bacterial SRP-mediated translocation pathway components have been identified. Biochemical analysis has revealed that although the archaeal system incorporates various facets of the eukaryal and bacterial targeting systems, numerous aspects of the archaeal system are unique to this domain of life. Moreover, it is becoming increasingly clear that elucidation of the archaeal SRP pathway will provide answers to basic questions about protein targeting that cannot be obtained from examination of eukaryal or bacterial models. In this review, recent data regarding the molecular composition, functional behavior and evolutionary significance of the archaeal signal recognition particle pathway are discussed.

Archaea↗

PubMed1996

Expression and purification of the canine 54-kDa subunit of signal recognition particle as a his-tagged protein from Escherichia coli.

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.

Amino Acid Sequence↗

PubMed1987

Mathematical modeling of the effects of the signal recognition particle on translation and translocation of proteins across the endoplasmic reticulum membrane.

The kinetics of the signal recognition particle(SRP)-mediated process of protein translocation across the endoplasmic reticulum membrane was studied by mathematical modeling and complementary experiments. The following results were obtained. (1) A model according to which SRP directs the ribosome, rather than the mRNA, to the membrane is supported by experiments designed to discriminate between the two alternatives. (2) This model describes both steady-state and synchronized translation experiments and makes a number of predictions. (3) The interaction between a nascent protein and SRP may be described by two parameters: (i) a binding constant which can be attributed to the structure of the signal peptide, and (ii) the size of the "SRP-window", i.e. the distance between the first and the last site on the polypeptide chain that can interact with SRP. For preprolactin a binding constant of 1 to 2.5 nmol-1l was estimated. Modeling of the synchronized synthesis of ovalbumin indicates that it has a much weaker binding constant than preprolactin (approximately 0.25 nmol-1l) although we cannot exclude the possibility that the SRP-window may be also smaller. (4) A better understanding of the molecular effects of SRP on translation and translocation through the rough endoplasmic reticulum membrane has been achieved. Inhibition of the steady-state rate of translation by SRP requires a stoichiometric interaction of SRP with ribosomes carrying nascent polypeptide chains and will occur only when ribosomes are piled up back to the initiation site. Translocation, on the other hand, requires only the catalytic action of SRP and is determined by the local concentration of protein-synthesizing ribosomes accumulated at the site(s) of SRP interaction. As a consequence, translational inhibition by SRP may sometimes fail to occur, depending either on the type of protein or on experimental conditions, such as a high mRNA concentration, even if translocation can be demonstrated. (5) A rough extrapolation to the conditions in vivo indicates that all synthesized polypeptide chains destined for translocation across or integration into the endoplasmic reticulum membrane are indeed quantitatively translocated and that no translational inhibition occurs.

Endoplasmic Reticulum↗

PubMed1997

Purification and biochemical characterization of the 19-kDa signal recognition particle RNA-binding protein expressed as a hexahistidine-tagged polypeptide in Escherichia coli.

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.

Amino Acid Sequence↗

PubMed1992

Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19.

The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.

Amino Acid Sequence↗

PubMed1998

Another factor besides hydrophobicity can affect signal peptide interaction with signal recognition particle.

Translocation of alkaline extracellular protease (AEP) into the endoplasmic reticulum of Yarrowia lipolytica is cotranslational and signal recognition particle (SRP)-dependent, whereas translocation of P17M AEP (proline to methionine at position 17, second amino acid in the pro-region) is posttranslational and SRP-independent. P17M signal peptide mutations that resulted in more rapid SRP-dependent translocation of AEP precursor were isolated. Most of these mutations significantly increased hydrophobicity, but the A12P/P17M mutation did not. The switch from SRP-dependent to SRP-independent translocation without a decrease in hydrophobicity (wild type to P17M) and restoration of SRP-dependent translocation without an increase in hydrophobicity (P17M to A12P/P17M) indicate that some factor(s) in addition to hydrophobicity determines selection of targeting pathway. Models of extended forms of wild type and A12P/P17M signal peptides are kinked, whereas the P17M signal peptide is relatively straight. Possibly the conformation/orientation of signal peptides at the ribosomal surface affects SRP binding and consequently the targeting route to the endoplasmic reticulum. Kinked signal peptides might approach SRP more closely more often. Most likely, these effects were only detectable because of the short length and low average hydrophobicity of the AEP signal peptide.

Ascomycota↗

PubMed2006

The structure of the mammalian signal recognition particle (SRP) receptor as prototype for the interaction of small GTPases with Longin domains.

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.

Affinity Labels↗

PubMed2002

Identification and characterization of Streptococcus pneumoniae Ffh, a homologue of SRP54 subunit of mammalian signal recognition particle.

Recent studies have demonstrated that bacteria possess an essential protein translocation system similar to mammalian signal recognition particle (SRP). Here we have identified the Ffh, a homologue of the mammalian SRP54 subunit from S. pneumoniae. Ffh is a 58-kDa protein with three distinct domains: an N-terminal hydrophilic domain (N-domain), a G-domain containing GTP/GDP binding motifs, and a C-terminal methionine-rich domain (M-domain). The full-length Ffh and a truncated protein containing N and G domains (Ffh-NG) were overexpressed in E. coli and purified to homogeneity. The full-length Ffh has an intrinsic GTPase activity with k(cat) of 0.144 min(-1), and the K(m) for GTP is 10.9 microM. It is able to bind to 4.5S RNA specifically as demonstrated by gel retardation assay. The truncated Ffh-NG has approximately the same intrinsic GTPase activity to the full-length Ffh, but is unable to bind to 4.5S RNA, indicating that the NG domain is sufficient for supporting intrinsic GTP hydrolysis, and that the M domain is required for RNA binding. The interaction of S. pneumoniae Ffh with its receptor, FtsY, resulted in a 20-fold stimulation in GTP hydrolysis. The stimulation was further demonstrated to be independent of the 4.5S RNA. In addition, a similar GTPase stimulation is also observed between Ffh-NG and FtsY, suggesting that the NG domain is sufficient and the M domain is not required for GTPase stimulation between Ffh and FtsY.

Amino Acid Sequence↗

PubMed1989

Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle.

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.

Amino Acid Sequence↗

PubMed1996

Crystallization and preliminary X-ray analysis of the 9 kDa protein of the mouse signal recognition particle and the selenomethionyl-SRP9.

Two different crystal forms of the 9 kDa protein of the signal recognition particle (SRP9) have been prepared by the hanging drop vapor diffusion technique using 28% (w/v) PEG8000 or 28% saturated ammonium sulphate as precipitant. The crystals are hexagonal bipyramids with average dimensions of 0.2 X 0.1 X 0.1 mm(3) and they diffract to a resolution of 2.3 Angstroms. They belong to the space groups P6(2)22/P6(4)22 or P3(1)21/P3(2)21 with cell dimensions a = b = 63.0 Angstroms, and c = 111.5 Angstroms. Crystals have also been grown from the selenomethionyl protein and multiwavelength data sets have been collected.

Animals↗

PubMed1990

Assembly of the Alu domain of the signal recognition particle (SRP): dimerization of the two protein components is required for efficient binding to SRP RNA.

The signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, plays an essential role in targeting secretory proteins to the rough endoplasmic reticulum membrane. In addition to the targeting function, SRP contains an elongation arrest or pausing function. This function is carried out by the Alu domain, which consists of two proteins, SRP9 and SRP14, and the portion of SRP (7SL) RNA which is homologous to the Alu family of repetitive sequences. To study the assembly pathway of the components in the Alu domain, we have isolated a cDNA clone of SRP9, in addition to a previously obtained cDNA clone of SRP14. We show that neither SRP9 nor SRP14 alone interacts specifically with SRP RNA. Rather, the presence of both proteins is required for the formation of a stable RNA-protein complex. Furthermore, heterodimerization of SRP9 and SRP14 occurs in the absence of SRP RNA. Since a partially reconstituted SRP lacking SRP9 and SRP14 [SRP(-9/14)] is deficient in the elongation arrest function, it follows from our results that both proteins are required to assemble a functional domain. In addition, SRP9 and SRP14 synthesized in vitro from synthetic mRNAs derived from their cDNA clones restore elongation arrest activity to SRP(-9/14).

Amino Acid Sequence↗

PubMed1987

Signal recognition particle arrests elongation of nascent secretory and membrane proteins at multiple sites in a transient manner.

The signal recognition particle (SRP) has been shown to target nascent secretory and membrane proteins to the endoplasmic reticulum. In the wheat germ cell-free system, SRP arrests the elongation of the nascent chains until the translational complex is docked to the endoplasmic reticulum membrane where the interaction between SRP and docking protein causes a release of the nascent chain arrest. For two secretory proteins, arrested peptides of 70 amino acids have been identified (Walter, P., Ibrahimi, I., and Blobel, G. (1981) J. Cell Biol. 91, 545-550; Meyer, D. I., Krause, E., and Dobberstein, B. (1982) Nature 297, 647-650). By using an in vitro coupled transcription-translation system, we have analyzed SRP arrest and the resulting peptides of the two secretory proteins lysozyme and granulocyte-macrophage colony-stimulating factor and the membrane protein invariant chain. SRP arrested the elongation of all three proteins at multiple sites, giving rise to ladders of arrested peptides. The size of the arrested peptides increased with the time of translation, resulting in mostly full-length pre-peptides after about 40 min. This suggests that SRP arrest in transient rather than stable. Upon addition of microsomes, the SRP arrest was released, and all the blocked peptides could be chased into mature proteins or full-length precursors.

Animals↗

PubMed2006

Anti-signal recognition particle autoantibodies: marker of a necrotising myopathy.

OBJECTIVE: To elucidate the clinical importance of the anti-signal recognition particle (SRP) autoantibody in patients with myositis. METHODS: Retrospective systematic assessment of the clinical, laboratory and histological characteristics of 23 anti-SRP-positive patients from six European centres. Data were compared with a large group of anti-SRP-negative patients with myositis published previously. RESULTS: Clinically, patients with anti-SRP autoantibodies often had a severe symmetric proximal muscle weakness resulting in marked disability, dysphagia and highly elevated levels of serum creatine kinase. Three patients had typical dermatomyositis rashes. The disease was associated with the occurrence of extramuscular signs and symptoms including interstitial lung disease. No association was found with an increased risk of cardiac involvement, and the disease carried a reasonably favourable prognosis with most patients responding to treatment. None of the patients had the typical histological features of myositis. Most muscle biopsy specimens showed the presence of necrotic muscle fibres and no inflammatory infiltrates. CONCLUSIONS: Anti-SRP autoantibodies are associated with a syndrome of a necrotising myopathy in the spectrum of immune-mediated myopathies that differs from typical polymyositis. Further studies are needed to elucidate the pathogenesis and to clarify the role of the anti-SRP autoantibodies in this unique disease.

Adult↗

PubMed2001

Purification, characterization, and cloning of the cDNA of human signal recognition particle RNA 3'-adenylating enzyme.

The 3'-terminal adenylic acid residue in several human small RNAs including signal recognition particle (SRP) RNA, nuclear 7SK RNA, U2 small nuclear RNA, and ribosomal 5S RNA is caused by a post-transcriptional adenylation event (Sinha, K., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). Using the Alu portion of the SRP RNA as a substrate in an in vitro adenylation assay, we purified an adenylating enzyme that adds adenylic acid residues to SRP/Alu RNA from the HeLa cell nuclear extract. All the peptide sequences obtained by microsequencing of the purified enzyme matched a unique human cDNA corresponding to a new adenylating enzyme having homologies to the well characterized mRNA poly(A) polymerase. The amino terminus region of the human SRP RNA adenylating enzyme showed approximately 75% homology to the amino terminus of the human mRNA poly(A) polymerase that includes the catalytic domain. The carboxyl terminus of the human SRP RNA adenylating enzyme showed less than 25% homology to the carboxyl terminus of poly(A) polymerase, which interacts with other factors and provides specificity. The SRP RNA adenylating enzyme is coded for by a gene located on chromosome 2 in contrast to the poly(A) polymerase gene, which is located on chromosome 14. A recombinant protein for the SRP RNA adenylating enzyme was prepared, and its activity was compared with the purified enzyme from HeLa cells. The data indicate that in addition to the SRP RNA adenylating enzyme, other factors may be required to carry out accurate 3'-end adenylation of SRP RNA.

Amino Acid Sequence↗

PubMed2003

Structure and function of the chloroplast signal recognition particle.

The targeting of proteins, including the insertion and translocation of proteins in or across membranes, is a fundamental process within a cell, and a variety of specialized mechanisms for protein transport have been developed during evolution. The signal recognition particle (SRP) is found in the cytoplasm of most, if not all, eukaryotes and prokaryotes where it plays a central role in the co-translational insertion of membrane proteins into the endoplasmic reticulum and plasma membrane, respectively. SRP is a ribonucleoprotein consisting of an RNA and at least one polypeptide of approximately 54 kDa (SRP54). Interestingly, chloroplasts contain a specialized type of signal recognition particle. Chloroplast SRP (cpSRP) contains a SRP54 homologue but differs strikingly from cytosolic SRP in various aspects of structure and function. In contrast to cytosolic SRP, it contains a novel protein subunit (cpSRP43) and lacks RNA. CpSRP is also distinctive in its ability to interact with its substrate, light-harvesting chlorophyll a/ b-binding protein, post-translationally. Furthermore, it is remarkable that the 54 kDa subunit of cpSRP is also involved in the co-translational transport of chloroplast-encoded thylakoid proteins, and is therefore able to switch between the co- and post-translational means of interaction with its respective substrate proteins.

Arabidopsis↗

PubMed1990

Antibody to signal recognition particle in polymyositis.

Using immunoprecipitation, we identified 13 patients with antibodies to the signal recognition particle (SRP) from a collection of sera representing 265 polymyositis/dermatomyositis (PM/DM) patients. Antibody reactivity with SRP was confirmed by enzyme-linked immunosorbent assay and immunoprecipitation with isolated dog pancreas SRP. The antibody was present in the serum of 4% of PM/DM patients, and 18% of PM/DM patients with anticytoplasmic antibodies other than anti-Jo-1, but not in patients with other conditions who had anticytoplasmic antibodies. Anti-SRP was associated with classic adult PM, and some of these cases were unusually severe and/or of rapid onset; it was not found in patients with overlap syndromes or with DM involvement. Unlike patients with antibodies to aminoacyl-transfer RNA synthetases, patients with anti-SRP had a low frequency of pulmonary fibrosis, as well as of arthritis and Raynaud's phenomenon. Anti-SRP antibodies may serve as a marker for a second, distinct subgroup of adult PM.

Adult↗