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Identification of chloroplast signal recognition particle RNA genes.

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts. In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome. The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43. An RNA component has not been identified and does not seem to be required for the post-translational cpSRP. The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified. Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible. We have analysed chloroplast genomes from 27 organisms. In higher plant chloroplasts, no SRP RNA genes were identified. However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene. These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP.

Amino Acid Sequence↗

Comparative analysis of tertiary structure elements in signal recognition particle RNA.

BACKGROUND: The signal recognition particle (SRP) is a ribonucleoprotein complex that associates with ribosomes to promote co-translational translocation of proteins across biological membranes. We have used comparative analysis of a large number of bacterial, archaeal, and eukaryotic SRP RNA sequences to derive shared tertiary SRP RNA structure elements. RESULTS: A representative three-dimensional model of the human SRP RNA is shown that includes single-stranded intrahelical and interhelical RNA loops and incorporates data from enzymatic and chemical modification, electron microscopy, and site-directed mutagenesis. Properties of the SRP RNA model are an overall extended dumbbell-shaped structure (260 A x 70 A) with a pseudoknot in the small SRP domain (a pairing of 12-UGGC-15 with 33-GCUA-36), and a tertiary interaction in the large SRP domain (198-GA-199 with 232-GU-233). CONCLUSIONS: The RNA 'knuckle' formed in helix 8 of SRP RNA appears to constitute the binding site for protein SRP54 or its bacterial equivalent, protein P48. A dynamic property of this feature may explain the hierarchial assembly of proteins SRP19 and SRP54 in the large SRP domain. Furthermore, the human SRP RNA model serves as a framework to understand details of the structure and function of SRP in all organisms and is presented to stimulate further experimentation in this area.

Animals↗

Isolation and characterization of an Arabidopsis thaliana gene for the 54 kDa subunit of the signal recognition particle.

The first step in the routing of newly synthesized proteins into the secretory pathway is the binding of the nascent signal sequence to the signal recognition particle. The mammalian signal recognition particle is a complex consisting of 6 proteins and a single 7S RNA molecule. Signal recognition particle-like complexes have been described from wheat and maize but none of the protein components have yet been described from any plant species. Here we report the cloning and characterization of an Arabidopsis thaliana gene encoding the 54 kDa protein subunit of the signal recognition particle. This is the first report of a SRP-54 sequence for any plant species and the first genomic sequence for any multicellular organism.

Amino Acid Sequence↗

The archaeal signal recognition particle: steps toward membrane binding.

Signal recognition particles and their receptors target ribosome nascent chain complexes of preproteins toward the protein translocation apparatus of the cell. The discovery of essential SRP components in the third urkingdom of the phylogenetic tree, the archaea (Woese, C. R., and Fox, G. E. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 5088-5090) raises questions concerning the structure and composition of the archaeal signal recognition particle as well as the functions that route nascent prepoly peptide chains to the membrane. Investigations of the archaeal SRP pathway could therefore identify novel aspects of this process not previously reported or unique to archaea when compared with the respective eukaryal and bacterial systems.

Archaea↗

The organization of the 7SL RNA in the signal recognition particle.

Digestion of the signal recognition particle (SRP) of dog pancreas with micrococcal nuclease results in the stepwise cleavage of the 300 nucleotide 7SL RNA moiety producing five major fragments approximately 220 (1), 150 (2), 72 (3), 62 (4) and 45 (5) nucleotides long. The RNA molecule is initially cut once yielding fragments 1 and 3. Further degradation releases fragments 2, 4 and 5. The introduction of the first nick into the 7SL RNA does not alter the structure nor the function of the SRP. Further degradation of the RNA results in disruption and loss of activity of the particle. The sequence of the RNA fragments shows that the nuclease causes discrete cuts in the RNA with minimal nibbling indicating that only few sites are accessible to the action of the enzyme. The five major products of nuclease digestion together span almost the entire length of the 7SL RNA. Nicking occurs mainly around the boundary region between the central S sequence and the flanking Alu sequences constituting the 7SL RNA (1). The S fragment is bound to the four largest polypeptides while the 5' and 3' Alu fragments are associated with the two smallest protein constituents of the SRP.

Animals↗

Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its receptor.

The signal recognition particle (SRP) directs signal sequence specific targeting of ribosomes to the rough endoplasmic reticulum. Displacement of the SRP from the signal sequence of a nascent polypeptide is a guanosine triphosphate (GTP)-dependent reaction mediated by the membrane-bound SRP receptor. A nonhydrolyzable GTP analog can replace GTP in the signal sequence displacement reaction, but the SRP then fails to dissociate from the membrane. Complexes of the SRP with its receptor containing the nonhydrolyzable analog are incompetent for subsequent rounds of protein translocation. Thus, vectorial targeting of ribosomes to the endoplasmic reticulum is controlled by a GTP hydrolysis cycle that regulates the affinity between the SRP, signal sequences, and the SRP receptor.

Animals↗

Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2.

The signal recognition particle (SRP)-translocation pathway is conserved in all three domains of life and delivers membrane and secretory proteins to the cytoplasmic membrane or endoplasmic reticulum. We determined the requirement in the cariogenic oral pathogen Streptocococcus mutans of the three universally conserved elements of the SRP pathway: Ffh/SRP54, scRNA, and FtsY/SRalpha. Previously, we reported that insertional interruption of S. mutans ffh was not lethal, but resulted in acid sensitivity. To test whether S. mutans could survive extensive disruption of the SRP pathway, single and double deletions of genes encoding Ffh, scRNA, and FtsY were generated. Without environmental stressors, all mutant strains were viable, but unlike the wild-type, none could initiate growth at pH 5.0 or in 3.5% NaCl. Survival of challenge with 0.3 mM H(2)O(2) was also diminished without ffh. Members of the YidC/Oxa1/Alb3 family are also ubiquitous, involved in the translocation and assembly of membrane proteins, and have been identified in prokaryotes/mitochondria/chloroplasts. Two genes encoding YidC homologs, YidC1 and YidC2, are present in streptococcal genomes with both expressed in S. mutans. Deletion of YidC1 demonstrated no obvious phenotype. Elimination of YidC2 resulted in a stress-sensitive phenotype similar to SRP pathway mutants. Mutants lacking both YidC2 and SRP components were severely impaired and barely able to grow, even in the absence of environmental stress. Here, we report the dispensability of the cotranslational SRP protein translocation system in a bacterium. In S. mutans, this pathway contributes to protection against rapid environmental challenge and may overlap functionally with YidC2.

Bacterial Proteins↗

The N-domain of the signal recognition particle 54-kDa subunit promotes efficient signal sequence binding.

The signal recognition particle 54-kDa subunit (SRP54) binds to the signal sequences of nascent presecretory and transmembrane proteins. Previous studies have shown that signal sequences bind to the C-terminal methionine-rich domain of the protein (M-domain), but have raised the possibility that either the N-terminal domain (N-domain) or the central guanosine triphosphatase module (GTPase-domain) also contribute to signal-sequence-binding activity. We have generated a series of N-domain and GTPase-domain mutants to investigate this issue further. Mutations in a conserved N-domain motif (ALLEADV) produced significant defects in signal sequence binding that correlate with the severity of the mutation. The magnitude of the defect was independent of the preprotein substrate, which suggested that the mutations do not alter the specificity of signal sequence recognition. The N-domain mutants also showed defects in promoting the translocation of presecretory proteins across the membrane of microsomal vesicles, but these defects appeared to be a direct consequence of the reduction in signal-sequence-binding activity and not separate effects of the mutations. By contrast, mutations in the guanosine triphosphatase consensus sequence had no effect on signal sequence binding, but instead severely impaired protein translocation activity. These results indicate that a principal function of the SRP54 N-domain is to promote efficient signal sequence binding. These data also suggest that the SRP54 GTPase regulates the cycle of signal sequence binding and release, perhaps by modulating the relative orientation of the N- and M-domains.

Animals↗

The 2 A structure of helix 6 of the human signal recognition particle RNA.

BACKGROUND: The mammalian signal recognition particle (SRP) is an essential cytoplasmic ribonucleoprotein complex involved in targeting signal-peptide-containing proteins to the endoplasmic reticulum. Assembly of the SRP requires protein SRP19 to bind first to helix 6 of the SRP RNA before the signal-peptide-recognizing protein, SRP54, can bind to helix 8 of the RNA. Helix 6 is closed by a GGAG tetraloop, which has been shown to form part of the SRP19-binding site. RESULTS: The high-resolution (2.0 A) structure of a fragment of human SRP RNA comprising 29 nucleotides of helix 6 has been determined using the multiple anomalous dispersion (MAD) method and bromine-labelled RNA. In the crystal the molecule forms 28-mer duplexes rather than the native monomeric hairpin structure, although two chemically equivalent 11 base pair stretches of the duplex represent the presumed native structure. The duplex has highly distorted A-RNA geometry caused by the occurrence of several non-Watson-Crick base pairs. These include a 5'-GGAG-3'/3'-GAGG-5' purine bulge (which replaces the tetraloop) and a 5'-AC-3'/3'-CA-5' tandem mismatch that, depending on the protonation state of the adenine bases, adopts a different conformation in the two native-like parts of the structure. The structure also shows the 2'3'-cyclic phosphate reaction product of the hammerhead ribozyme cleavage reaction. CONCLUSIONS: The 29-mer RNA is the first RNA structure of the human SRP and provides some insight into the binding mode of SRP19. The observed strong irregularities of the RNA helix make the major groove wide enough and flat enough to possibly accommodate an alpha helix of SRP19. The variety of non-canonical base pairs observed enlarges the limited repertoire of irregular RNA folds known to date and the observed conformation of the 2'3'-cyclic phosphate containing Ade29 is consistent with the current understanding of the hammerhead ribozyme reaction mechanism.

Base Sequence↗

Distinct modes of signal recognition particle interaction with the ribosome.

Signal recognition particle (SRP), together with its receptor (SR), mediates the targeting of ribosome-nascent chain complexes to the endoplasmic reticulum. Using protein cross-linking, we detected distinct modes in the binding of SRP to the ribosome. During signal peptide recognition, SRP54 is positioned at the exit site close to ribosomal proteins L23a and L35. When SRP54 contacts SR, SRP54 is rearranged such that it is no longer close to L23a. This repositioning may allow the translocon to dock with the ribosome, leading to insertion of the signal peptide into the translocation channel.

Animals↗

Crystallization and preliminary x-ray diffraction studies on the conserved GTPase domain of the signal recognition particle from Acidianus ambivalens.

The signal recognition particle (SRP) of bacteria consists of only one protein, known as Ffh or the SRP54 homologue, which forms a complex with 4.5S RNA. It also binds to signal peptides and contains a GTPase which displays interesting differences to Ras GTPases. The conserved NG-domain of Ffh from the archaebacterium Acidianus ambivalens was cloned and overexpressed with a C-terminal His tag in Escherichia coli. Crystallization experiments of the native protein as well as of the Thr112Ala mutant, which is deficient in GTP hydrolysis, resulted in crystals suitable for X-ray diffraction. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 64.5, b = 128.3, c = 72.0 A. At cryogenic temperatures, the crystals diffracted to a resolution limit of 2.8 A using a rotating-anode generator and contain one molecule per asymmetric unit. A native data set has been collected using synchrotron radiation to around 2.0 A resolution. Selenomethionine protein was produced; its crystals diffract in-house to about 2.8 A resolution.

Archaea↗

Advances in the structure and functions of signal recognition particle in protein targeting.

The signal recognition particle (SRP) is a unique moiety in living cells, which has been conserved during evolution for protein targeting and translocation across membranes in collaboration with its receptor (SR). The structural and functional features of its components, (six polypeptides and RNA) are being rapidly elucidated. We have endeavored in this review to epitomize most recent advances in this field. Its two domains (S and Alu) play important roles in signal recognition, elongation arrest and protein targeting of the polypeptide being synthesized in the cytoplasm. SRP14 and SRP9 help in the elongation arrest by interacting with signal peptide. GTPase activity of SRP54 releases SRP from SR. In addition, alpha and beta subunits of SR also possess GTPase activities and the three GTPases help in docking of nascent peptide chain-ribosome complex to the translocation site. Further strides in proteomics employing mass spectrometry and X-ray crystallography are expected to throw more light on the molecular events occurring during protein targeting and translocation.

Animals↗

The signal recognition particle database (SRPDB).

The SRPDB (signal recognition particle database) provides aligned SRP RNA and protein sequences, annotated and phylogenetically ordered. This release includes 82 SRP RNAs (including 22 bacterial and 9 archaeal homologs) and a total of 20 protein sequences representing SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72. The offerings also include representative RNA secondary structure diagrams.

Amino Acid Sequence↗

The signal recognition particle database (SRPDB).

The SRPDB (signal recognition particle database) provides annotated SRP RNA sequences from Eucaryotes and Archaea, phylogenetically ordered and aligned with their bacterial equivalents. We also make available representative RNA secondary structure diagrams, where each base pair is proven by comparative sequence analysis. New to this release are 17 SRP RNA sequences (a total of 64 sequences) and alignments of proteins SRP19 and SRP54 with their RNA binding sites.

Animals↗

Signal recognition particle (SRP), a ubiquitous initiator of protein translocation.

In higher eukaryotes, most secretory and membrane proteins are synthesised by ribosomes which are attached to the membrane of the rough endoplasmic reticulum (RER). This allows the proteins to be translocated across that membrane already during their synthesis. The ribosomes are directed to the RER membrane by a cytoplasmic ribonucleoprotein particle, the signal recognition particle (SRP). SRP fulfills its task by virtue of three distinguishable activities: the binding of a signal sequence which, being part of the nascent polypeptide to be translocated, is exposed on the surface of a translating ribosome; the retardation of any further elongation; and the SRP-receptor-mediated binding of the complex of ribosome, nascent polypeptide and SRP to the RER membrane which results in the detachment of SRP from the signal sequence and the ribosome and the insertion of the nascent polypeptide into the membrane. Evidence is accumulating that SRP is not restricted to eukaryotes: SRP-related particles and SRP-receptor-related molecules are found ubiquitously and may function in protein translocation in every living organism. This review focuses on the mammalian SRP. A brief discussion of its overall structure is followed by a detailed description of the structures of its RNA and protein constituents and the requirements for their assembly into the particle. Homologues of SRP components from organisms other than mammals are mentioned to emphasize the components' conserved or less conserved features. Subsequently, the functions of each of the SRP constituents are discussed. This sets the stage for a presentation of a model for the mechanism by which SRP cyclically assembles and disassembles with translating ribosomes and the RER membrane. It may be expected that similar mechanisms are used by SRP homologues in organisms other than mammals. However, the mammalian SRP-mediated translocation mechanism may not be conserved in its entirety in organisms like Escherichia coli whose SRP lack components required for the function of the mammalian SRP. Possible translocation pathways involving the rudimentary SRP are discussed in view of the existence of alternative, chaperone-mediated translocation pathways with which they may intersect. The concluding two sections deal with open questions in two areas of SRP research. One formulates basic questions regarding the little-investigated biogenesis of SRP. The other gives an outlook over the insights into the mechanisms of each of the known activities of the SRP that are to be expected in the short and medium-term future.

Animals↗

Cooperative assembly of signal recognition particle RNA with protein SRP19.

Signal recognition particle (SRP) is a ribonucleoprotein complex involved in the targeting of secretory proteins to the lipid bilayer of the endoplasmic reticulum. SRP contains the protein SRP19, which is an important structural and functional component, believed to promote the assembly of the particle. We have purified the human SRP19 protein to homogeneity from recombinant bacteria which overexpress the polypeptide, and have studied details of the binding to SRP RNA via gel mobility shift and RNase sensitivity assays. SRP19 interacts with two SRP RNA conformers with different affinities such that the more compact RNA species is bound more avidly. Furthermore, binding was found to be highly cooperative. Binding constants and Hill coefficients were determined for several mutant SRP RNAs in which individual RNA helices were deleted. These results confirmed that both SRP RNA helices 6 and 8 are important for SRP19 binding. Enzymatic RNA structure probing of a 150-nucleotide mutant SRP RNA fragment and of the corresponding RNA-SRP19 complex showed that cooperativity may be due to protein-induced conformational changes in the large domain of the SRP RNA. Finally, SRP19 bound specifically not only to SRP RNA but also to the A-form of Escherichia coli 5S ribosomal RNA, thereby indicating structural similarities between these two RNA molecules.

Base Sequence↗

The Signal Recognition Particle Database (SRPDB).

The SRPDB (Signal Recognition Particle Database) offers aligned SRP RNA and SRP protein sequences, phylogenetically ordered and annotated. This release adds three SRP RNA sequences (totaling 96 SRP RNA sequences) and 11 SRP protein sequences (a total of 39 protein sequences from SRP9, SRP14, SRP19, SRP21, SRP54, SRP68 or SRP72). Also downloadable are sample SRP RNA secondary structure diagrams, a three-dimensional model of the human SRP RNA, search motifs and software.

Amino Acid Sequence↗

The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences.

The signal recognition particle (SRP) binds to signal sequences when they emerge from a translating ribosome and targets the complex of ribosome, nascent chain and SRP to the membrane of the rough endoplasmic reticulum (rER) allowing the co-translational translocation of the nascent chain. By photo-crosslinking it has been shown that the signal sequence of preprolactin (PPL) only interacts with the methionine-rich (M) domain of the 54 kDa protein subunit (SRP54) of SRP. Here we show that (i) a signal-anchor sequence is likewise crosslinked only to the methionine-rich domain of SRP54, (ii) free SRP54 can interact with signal sequences independently of the other components of SRP, (iii) its M domain suffices to perform this function, and (iv) an essentially intact M domain is required for signal sequence recognition. Alkylation of the N+G domain in intact SRP54 with N-ethyl maleimide (NEM), but not after cleavage with V8 protease, prevents the binding of a signal sequence to the M domain. This suggests a proximity between the N+G and M domains of SRP54 and raises the possibility that the role of the N+G domain may be to regulate the binding and/or the release of signal sequences.

Animals↗