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Results for “Signal Recognition Particle”

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PubMed1993

GTPase activity of a bacterial SRP-like complex.

We have recently identified a protein (SRPM54) in Mycoplasma mycoides homologous to SRP54, a subunit of the mammalian signal recognition particle (SRP). This protein forms a complex with a mycoplasma RNA related to the RNA component of SRP. We have now demonstrated that the protein has an intrinsic GTPase activity in vitro and kinetic parameters for the enzymatic reaction have been determined. The GTPase activity was not significantly affected by the presence of the mycoplasma SRP RNA. Different regions of the SRPM54 protein were expressed as recombinant proteins in E. coli and were purified to near homogeneity. On the basis of the properties of these SRPM54 fragments two different functional domains of the protein could be distinguished. An N-terminal part was found to contain the GTPase activity and this domain had approximately the same kinetic properties as the full-length protein. Another domain corresponding to a C-terminal fragment contained the RNA binding activity as shown using an assay based on the retention of RNA-protein complexes to nitrocellulose filters.

Bacterial Proteins↗

PubMed1998

Heterodimer SRP9/14 is an integral part of the neural BC200 RNP in primate brain.

BC200 RNA is a brain-specific, small non-messenger RNA with a somatodendritic localization in primate neurons and a constituent of a ribonucleoprotein (RNP) complex. The primary and secondary structure of the 5' domain of BC200 RNA resembles that of the Alu domain of 7SL RNA, which is an integral part of the signal recognition particle (SRP). This would predict that similar proteins bind to this defined domain of both RNA species in vitro and in vivo. The data presented in this paper reveal that a protein that binds BC200 RNA in vivo is immunoreactive with antibodies against SRP9. This further supports the notion that the 5' domain of the BC200 RNA can fold into structures similar to the SRP Alu domain and, as a result, bind identical or similar proteins in vivo. The SRP9 protein binds only as dimer with SRP14 protein to the Alu domain of 7SL RNA to form a subdomain that, in SRP, is functional in translation arrest. Therefore, our data also indicate that the neuronal BC200 RNP is a candidate for regulating decentralized protein biosynthesis in dendrites, possibly with a mechanism that resembles translation arrest of the SRP.

Animals↗

PubMed2002

Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act'.

The signal recognition particle (SRP) in bacteria and endoplasmic reticulum is involved in co-translational targeting. Plastids contain cpSRP54 and cpSRP43, unique to plants, but lack a SRP RNA molecule. A role for cpSRP in biogenesis of plastid-encoded membrane proteins has not been firmly established yet. In this study, a transient interaction between cpSRP54 and elongating D1 protein was observed using a homologous chloroplast translation system. Using the novel approach of cross-linking at different time points during elongation of full-length D1 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain forming two distinct cross-linked products. However, this interaction did not lead to an elongation arrest and cpSRP54 was released from the nascent chains, once they were longer than approximately 14 kDa. Detailed mutant analysis showed that the cpSRP54 interaction occurred via the first transmembrane domain, which could be replaced by other hydrophobic domains of more than 10 amino acids.

Chloroplasts↗

PubMed2006

The mechanosensitive channel protein MscL is targeted by the SRP to the novel YidC membrane insertion pathway of Escherichia coli.

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypo-osmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle (SRP) for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon. Mutants affected in the Sec-components are competent for MscL assembly. Translocation of the periplasmic domain was detected using a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The modification of a single cysteine residue at position 68 indicated its translocation across the inner membrane. From these in vivo experiments, it is concluded that the electrical chemical membrane potential is not necessary for membrane insertion of MscL. However, depletion of the membrane insertase YidC inhibits translocation of the protein across the membrane. We show here that YidC is essential for efficient membrane insertion of the MscL protein. YidC is a component of a recently identified membrane insertion pathway that is evolutionarily conserved in bacteria, mitochondria and chloroplasts.

Adenosine Triphosphatases↗

PubMed2001

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA.

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.

Amino Acid Sequence↗

PubMed2003

Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids: substrate not required.

Integration of thylakoid proteins by the chloroplast signal recognition particle (cpSRP) posttranslational transport pathway requires the cpSRP, an SRP receptor homologue (cpFtsY), and the membrane protein ALB3. Similarly, Escherichia coli uses an SRP and FtsY to cotranslationally target membrane proteins to the SecYEG translocase, which contains an ALB3 homologue, YidC. In neither system are the interactions between soluble and membrane components well understood. We show that complexes containing cpSRP, cpFtsY, and ALB3 can be precipitated using affinity tags on cpSRP or cpFtsY. Stabilization of this complex with GMP-PNP specifically blocks subsequent integration of substrate (light harvesting chl a/b-binding protein [LHCP]), indicating that the complex occupies functional ALB3 translocation sites. Surprisingly, neither substrate nor cpSRP43, a component of cpSRP, was necessary to form a complex with ALB3. Complexes also contained cpSecY, but its removal did not inhibit ALB3 function. Furthermore, antibody bound to ALB3 prevented ALB3 association with cpSRP and cpFtsY and inhibited LHCP integration suggesting that a complex containing cpSRP, cpFtsY, and ALB3 must form for proper LHCP integration.

Arabidopsis↗

PubMed2004

Methionine sulfoxide reductases protect Ffh from oxidative damages in Escherichia coli.

In proteins, methionine residues are primary targets for oxidation. Methionine oxidation is reversed by methionine sulfoxide reductases A and B, a class of highly conserved enzymes. Ffh protein, a component of the ubiquitous signal recognition particle, contains a methionine-rich domain, interacting with a small 4.5S RNA. In vitro analyses reported here show that: (i) oxidized Ffh is unable to bind 4.5S RNA, (ii) oxidized Ffh contains methionine sulfoxide residues, (iii) oxidized Ffh is a substrate for MsrA and MsrB enzymes; and (iv) MsrA/B repairing activities allow oxidized Ffh to recover 4.5S RNA-binding abilities. In vivo analyses reveal that: (i) Ffh synthesized in the msrA msrB mutant contains methionine sulfoxide residues and is unstable, (ii) msrA msrB mutant requires high levels of Ffh synthesis for growth and (iii) msrA msrB mutation leads to defects in Ffh-dependent targeting of MalF. We conclude that MsrA and MsrB are required to repair Ffh oxidized by reactive oxygen species produced by aerobic metabolism, establishing an as-yet undescribed link between protein targeting and oxidation.

Amino Acid Sequence↗

PubMed2003

Induced nucleotide specificity in a GTPase.

In signal-recognition particle (SRP)-dependent protein targeting to the bacterial plasma membrane, two GTPases, Ffh (a subunit of the bacterial SRP) and FtsY (the bacterial SRP receptor), act as GTPase activating proteins for one another. The molecular mechanism of this reciprocal GTPase activation is poorly understood. In this work, we show that, unlike other GTPases, free FtsY exhibits only low preference for GTP over other nucleotides. On formation of the SRP.FtsY complex, however, the nucleotide specificity of FtsY is enhanced 10(3)-fold. Thus, interactions with SRP must induce conformational changes that directly affect the FtsY GTP-binding site: in response to SRP binding, FtsY switches from a nonspecific "open" state to a "closed" state that provides discrimination between cognate and noncognate nucleotides. We propose that this conformational change leads to more accurate positioning of the nucleotide and thus could contribute to activation of FtsY's GTPase activity by a novel mechanism.

Bacterial Proteins↗

PubMed1994

Structural requirements of Bacillus subtilis small cytoplasmic RNA for cell growth, sporulation, and extracellular enzyme production.

Bacillus subtilis small cytoplasmic RNA (scRNA; 271 nucleotides) is a member of the signal recognition particle (SRP) RNA family, which has evolutionarily conserved primary and secondary structures. The scRNA consists of three domains corresponding to domains I, II, and IV of human SRP 7S RNA. To identify the structural determinants required for its function, we constructed mutant scRNAs in which individual domains or conserved nucleotides were deleted, and their importance was assayed in vivo. The results demonstrated that domain IV of scRNA is necessary to maintain cell viability. On the other hand, domains I and II were not essential for vegetative growth but were preferentially required for the RNA to achieve its active structure, and assembled ribonucleoprotein between Ffh and scRNA is required for sporulation to proceed. This view is highly consistent with the fact that the presence of domains I and II is restricted to sporeforming B. subtilis scRNA among eubacterial SRP RNA-like RNAs.

Bacillus subtilis↗

PubMed1999

Analysis of Escherichia coli 4.5S RNA binding affinity to Ffh and EF-G.

Escherichia coli 4.5S RNA is a member of the signal recognition particle RNA family that binds to Ffh and EF-G proteins in vivo. To assess the binding affinity of E. coli 4.5S RNA, wild-type Ffh and a series of amino terminal truncated EF-G mutants with a histidine tag were over-expressed in Escherichia coli and purified. Among them, EF-G mutants with a deletion of all upstream sequences up to and including the second or the third GTP binding sequence element were expressed at high levels and bound with the same activity as wild-type EF-G. Nitrocellulose filter binding assays revealed that the binding affinity values (M(1/2)) for Ffh and EF-G, defined as the concentration giving half-maximal binding, were 0.15 microM and 1.5 microM, respectively. Moreover, we also show that very little EF-G can form a stable complex with 4.5S RNA in vivo, whereas almost all Ffh binds to 4.5S RNA.

Amino Acid Sequence↗

PubMed2000

Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon.

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY-SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.

Adenosine Triphosphatases↗

PubMed2003

RNA polymerase III transcripts and the PTB protein are essential for the integrity of the perinucleolar compartment.

The perinucleolar compartment (PNC) is a nuclear substructure present in transformed cells. The PNC is defined by high concentrations of certain RNA binding proteins and a subset of small RNAs transcribed by RNA polymerase III (pol III), including the signal recognition particle RNA and an Alu RNA as reported here. To determine if the PNC is dependent on pol III transcription, HeLa cells were microinjected with the selective pol III inhibitor, Tagetin. This resulted in disassembly of the PNC, whereas inhibition of pol I by cycloheximide or pol II by alpha-amanitin did not significantly affect the PNC. However, overexpression of one of the PNC-associated RNAs from a pol II promoter followed by injection of Tagetin blocked the Tagetin-induced PNC disassembly, demonstrating that it is the RNA rather than pol III activity that is important for the PNC integrity. To elucidate the role of the PNC-associated protein PTB, its synthesis was inhibited by siRNA. This resulted in a reduction of the number of PNC-containing cells and the PNC size. Together, these findings suggest, as a working model, that PNCs may be involved in the metabolism of specific pol III transcripts in the transformed state and that PTB is one of the key elements mediating this process.

Alu Elements↗

PubMed1992

A mutant of 7SL RNA in Yarrowia lipolytica affecting the synthesis of a secreted protein.

The yeast Yarrowia lipolytica contains two genes, SCR1 and SCR2, encoding a 7SL RNA associated with a signal recognition particle (SRP). To study 7SL RNA function in vivo we have systematically substituted the two conserved nucleotides G128 and A130 in loop 1 of SCR2-encoded 7SL RNA. All single mutations in either nucleotide have no effect. All double mutations are lethal except for one which results in temperature-sensitive growth. We have studied the synthesis and secretion of an alkaline extracellular protease (AEP) in both wild-type and temperature-sensitive mutant strains. Pulse-chase labeling and immunoprecipitation of this protein show that: 1) total protein synthesis is not affected in the mutant strain; 2) levels of AEP precursors in the mutant strain are 60% less than in the wild-type strain; 3) for both strains there is no accumulation of the AEP precursors in the cytoplasm; 4) the kinetics of secretion is similar. These results suggest that mutated SRP is deficient in membrane targeting but still performs translational arrest. This is consistent with the functions of SRP observed in vitro and represents the first demonstration of such roles in vivo.

Base Sequence↗

PubMed2005

Molecular crosstalk between the nucleotide specificity determinant of the SRP GTPase and the SRP receptor.

In signal recognition particle (SRP)-dependent targeting of proteins to the bacterial plasma membrane, two GTPases, Ffh (the SRP GTPase) and FtsY (the receptor GTPase), form a complex in which both proteins reciprocally stimulate each other's GTPase activities. We mutated Asp251 in the Ffh active site to Asn (D251N), converting Ffh to a xanthosine 5'-triphosphate (XTP)-specific protein as has been observed in many other GTPases. Unexpectedly, mutant SRP(D251N) is severely compromised in the formation of an active SRP.FtsY complex when bound with cognate XTP, and even more surprisingly, mutant SRP(D251N) works better when bound with noncognate GTP. These paradoxical results are explained by a model in which Ffh Asp251 forms a bidentate interaction with not only the bound GTP but also the receptor FtsY across the dimer interface. These interactions form part of the network that seals the lateral entrance to the composite active site at the dimer interface, thereby ensuring the electrostatic and/or structural integrity of the active site and contributing to the formation of an active SRP.FtsY complex.

Amino Acid Substitution↗

PubMed2003

Structure of an RNA dodecamer containing a fragment from SRP domain IV of Escherichia coli.

The crystal structure of an RNA dodecamer, r(GCGUCAGGUC(Br)CG)/r(CGGAAGCAG(Br)CGC), containing a fragment from the signal recognition particle (SRP) RNA (domain IV) of Escherichia coli, has been determined at 1.7 A resolution with 21 666 independent reflections and an R(work) and R(free) of 20.1 and 22.5%, respectively. The structure exhibits a novel crystal packing pattern for RNA oligomer duplexes: one end of the duplex adopts the stacking interaction, while the other end adopts the abutting interaction in the minor groove. The symmetric loop of the SRP, r(CAGG)/r(AGCA), in the center of the dodecamer forms two different conformations of the A.C mismatch, a sheared G.G and a symmetrical G.A mismatch. These four mismatches present a unique surface for the abutting interaction. The involvement of the two A.C mismatches in the abutting interaction implies that these mismatches are the important sites for interaction with proteins. The conformation of the symmetric loop is greatly stabilized by hydrated metal ions, which display flexibility in adjusting their geometry and coordination in interaction with nucleic acids. Comparison with other crystal structures of fragments of 4.5S RNA indicates that the conformation of the symmetric loop is independent of the asymmetrical loop in domain IV.

Crystallization↗

PubMed1997

The decline in human Alu retroposition was accompanied by an asymmetric decrease in SRP9/14 binding to dimeric Alu RNA and increased expression of small cytoplasmic Alu RNA.

Alu interspersed elements are inserted into the genome by a retroposition process that occurs via dimeric Alu RNA and causes genetic disorders in humans. Alu RNA is labile and can be diverted to a stable left monomer transcript known as small cytoplasmic Alu (scAlu) RNA by RNA 3' processing, although the relationship between Alu RNA stability, scAlu RNA production, and retroposition has been unknown. In vivo, Alu and scAlu transcripts interact with the Alu RNA-binding subunit of signal recognition particle (SRP) known as SRP9/14. We examined RNAs corresponding to Alu sequences that were differentially active during primate evolution, as well as an Alu RNA sequence that is currently active in humans. Mutations that accompanied Alu RNA evolution led to changes in a conserved structural motif also found in SRP RNAs that are associated with thermodynamic destabilization and decreased affinity of the Alu right monomer for SRP9/14. In contrast to the right monomer, the Alu left monomer maintained structural integrity and high affinity for SRP9/14, indicating that scAlu RNA has been under selection during human evolution. Loss of Alu right monomer affinity for SRP9/14 is associated with scAlu RNA production from Alu elements in vivo. Moreover, the loss in affinity coincided with decreased rates of Alu amplification during primate evolution. This indicates that stability of the Alu right monomer is a critical determinant of Alu retroposition. These results provide insight into Alu mobility and evolution and into how retroposons may interact with host proteins during genome evolution.

3T3 Cells↗

PubMed2005

Identification of an RNA-binding domain in human SRP72.

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.

Amino Acid Motifs↗

PubMed2000

Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity.

FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.

Anions↗