Evolutionary conserved nucleotides within the E.coli 4.5S RNA are required for association with P48 in vitro and for optimal function in vivo.

E.coli 4.5S RNA is homologous to domain IV of eukaryotic SPR7S RNA, the RNA component of the signal recognition particle. The 4.5S RNA is associated in vivo with a 48kD protein (P48), which is homologous to a protein component of the signal recognition particle, SRP54. In addition to secondary structural features, a number of nucleotides are conserved between the 4.5S RNA and domain IV of all other characterised SRP-like RNAs from eubacteria, arachaebacteria and eukaryotes. This domain consists of an extended stem-loop structure; conserved nucleotides lie within the terminal loop and within single-stranded regions bulged from the stem immediately preceding the loop. This conserved region is a candidate for the SRP54/P48 binding site. To determine the functional importance of this region within the 4.5S RNA, mutations were introduced into the 4.5S RNA coding sequence. Mutated alleles were tested for their function in vivo and for the ability of the corresponding RNAs to bind P48 in vitro. Single point mutations in conserved nucleotides within the terminal tetranucleotide loop do not affect P48 binding in vitro and produce only slight growth defects. This suggests that the sequence of the loop may be important for the structure of the molecule rather than for specific interactions with P48. On the other hand, nucleotides within the single-stranded regions bulged from the stem were found to be important both for the binding of P48 to the RNA and for optimal function of the RNA in vivo.     

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.     

Identification of RNA sequences and structural elements required for assembly of fission yeast SRP54 protein with signal recognition particle RNA.

Signal recognition particle (SRP) is a ribonucleoprotein composed of six polypeptides and a single RNA molecule. SRP RNA can be divided into four structural domains, the last of which is the most highly conserved and, in Schizosaccharomyces pombe, is the primary location to which deleterious mutations map. The ability of mammalian SRP54 protein (SRP54p) to bind Escherichia coli 4.5S RNA, a homolog of SRP RNA which contains only domain IV, suggested that SRP54p might interact directly with this region. To determine whether domain IV is critical for SRP54p binding in fission yeast cells, we used a native immunoprecipitation-RNA sequencing assay to test 13 mutant SRP RNAs for the ability to associate with the protein in vivo. The G156A mutation, which alters the 5' residue of the noncanonical first base pair of the domain IV terminal helix and confers a mild conditional growth defect, reduces assembly of the RNA with SRP54p. Mutating either of the two evolutionarily invariant residues in the bulged region 5' to G156 is more deleterious to growth and virtually abolishes SRP54p binding. We conclude that the conservation of nucleotides 154 to 156 is likely to be a consequence of their role as a sequence-specific recognition element for the SRP54 protein. We also tested a series of mutants with nucleotide substitutions in the conserved tetranucleotide loop and adjoining stem of domain IV. Although tetraloop mutations are deleterious to growth, they have little effect on SRP54p binding. Mutations which disrupt the base pair flanking the tetraloop result in conditional growth defects and significantly reduce association with SRP54p. Disruption of the other two base pairs in the short stem adjacent to the tetranucleotide loop has similar but less dramatic effects on SRP54p binding. These data provide the first evidence that both sequence-specific contacts and the structural integrity of domain IV of SRP RNA are important for assembly with SRP54p.     

Structure of 4.5S RNA in the signal recognition particle of Escherichia coli as studied by enzymatic and chemical probing.

The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in the conserved apical tetraloop are not protected. Hydroxyl radical treatment reveals an asymmetric pattern of backbone reactivity; in particular, the region encompassing nucleotides 60-82, i.e., the 3' part of the conserved domain IV, is protected. The data suggest that a bend in the domain IV region, most likely at the central asymmetric internal loop, is an important element of the tertiary structure of 4.5S RNA. Hyperchromicity and lead cleavage data are consistent with the model as they reveal the unfolding of a higher-order structure between 30 and 40 degrees C. Protection by protein P48 occurs in this region of the RNA and, more strongly, in the 5' part of domain IV (nt 26-50, most strongly from 35 to 49). It is likely that P48 binds to the outside of the bent form of 4.5S RNA.     

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.     

Structure of the most conserved internal loop in SRP RNA.

The signal recognition particle (SRP) directs translating ribosomes to the protein translocation apparatus of endoplasmic reticulum (ER) membrane or the bacterial plasma membrane. The SRP is universally conserved, and in prokaryotes consists of two essential subunits, SRP RNA and SRP54, the latter of which binds to signal sequences on the nascent protein chains. Here we describe the solution NMR structure of a 28-mer RNA composing the most conserved part of SRP RNA to which SRP54 binds. Central to this function is a six-nucleotide internal loop that assumes a novel Mg2+-dependent structure with unusual cross-strand interactions; besides a cross-strand A/A stack, two guanines form hydrogen bonds with opposite-strand phosphates. The structure completely explains the phylogenetic conservation of the loop bases, underlining its importance for SRP54 binding and SRP function.     

Structural insights into SRP RNA: an induced fit mechanism for SRP assembly

Proper assembly of large protein-RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein-RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-A resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.     

Induced structural changes of 7SL RNA during the assembly of human signal recognition particle

The eukaryotic signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein particle that targets secretory and membrane proteins to the endoplasmic reticulum. The binding of SRP54 to the S domain of 7SL RNA is highly dependent on SRP19. Here we present the crystal structure of a human SRP ternary complex consisting of SRP19, the M domain of SRP54 and the S domain of 7SL RNA. Upon binding of the M domain of SRP54 to the 7SL RNA-SRP19 complex, the asymmetric loop of helix 8 in 7SL RNA collapses. The bases of the four nucleotides in the long strand of the asymmetric loop continuously stack and interact with the M domain, whereas the two adenines in the short strand flip out and form two A-minor motifs with helix 6. This stabilizing interaction is only possible when helix 6 has been positioned parallel to helix 8 by the prior binding of SRP19 to the tetraloops of helices 6 and 8. Hence, the crystal structure of the ternary complex suggests why SRP19 is necessary for the stable binding of SRP54 to the S domain RNA.     

NMR structures of loop B RNAs from the stem-loop IV domain of the enterovirus internal ribosome entry site: a single C to U substitution drastically changes the shape and flexibility of RNA

The 5'-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem-loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5'-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5'-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain-RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition.     

Structures of SRP54 and SRP19, the two proteins that organize the ribonucleic core of the signal recognition particle from Pyrococcus furiosus

In all organisms the Signal Recognition Particle (SRP), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu). The 2.5 A resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely alpha-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 A resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19*SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP.     

Structure of a GDP:AlF4 complex of the SRP GTPases Ffh and FtsY, and identification of a peripheral nucleotide interaction site

The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF4. This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.     

RNA aptamers targeted to domain II of hepatitis C virus IRES that bind to its apical loop region

The internal ribosome entry site (IRES) is important for translation of hepatitis C virus (HCV) mRNA and has a unique RNA structure containing conserved domains I to IV. To investigate the function of domain II, we selected RNA aptamers that bind to domain II of HCV IRES by applying a simple and convenient selection method using a hybridized tag for fixing domain II RNA on magnetic beads instead of synthesizing long RNA. In addition, we employed surface plasmon resonance (SPR) technology to measure the binding affinity of each generation and to obtain detailed kinetic constants. The selected aptamers have a consensus sequence, 5'-UAUGGCU-3', which is complementary to the apical loop of domain II. The loop-loop interaction between the consensus sequence and domain II was confirmed by mutagenesis and nuclease mapping analyses. Binding affinities were dependent on the local structure containing the conserved sequence. The aptamers could inhibit IRES-dependent translation.     

Molecular Mechanism of GTPase Activation at the Signal Recognition Particle (SRP) RNA Distal End

The signal recognition particle (SRP) RNA is a universally conserved and essential component of the SRP that mediates the co-translational targeting of proteins to the correct cellular membrane. During the targeting reaction, two functional ends in the SRP RNA mediate distinct functions. Whereas the RNA tetraloop facilitates initial assembly of two GTPases between the SRP and SRP receptor, this GTPase complex subsequently relocalizes ∼100 Å to the 5′,3′-distal end of the RNA, a conformation crucial for GTPase activation and cargo handover. Here we combined biochemical, single molecule, and NMR studies to investigate the molecular mechanism of this large scale conformational change. We show that two independent sites contribute to the interaction of the GTPase complex with the SRP RNA distal end. Loop E plays a crucial role in the precise positioning of the GTPase complex on these two sites by inducing a defined bend in the RNA helix and thus generating a preorganized recognition surface. GTPase docking can be uncoupled from its subsequent activation, which is mediated by conserved bases in the next internal loop. These results, combined with recent structural work, elucidate how the SRP RNA induces GTPase relocalization and activation at the end of the protein targeting reaction.     

Two strategically placed base pairs in helix 8 of mammalian signal recognition particle RNA are crucial for the SPR19-dependent binding of protein SRP54

Signal recognition particle (SRP) guides secretory proteins to biological membranes in all organisms. Assembly of the large domain of mammalian SRP requires binding of SRP19 prior to the binding of protein SRP54 to SRP RNA. The crystal structure of the ternary complex reveals the parallel arrangement of RNA helices 6 and 8, a bridging of the helices via a hydrogen bonded A149-A201 pair and protein SRP19, and two A minor motifs between the asymmetric loop of helix 8 (A213 and A214) and helix 6. We investigated which residues in helix 8 are responsible for the SRP19-dependent binding of SRP54 by taking advantage of the finding that binding of human SRP54 to Methanococcus jannaschii SRP RNA is independent of SRP19. Chimeric human/M. jannaschii SRP RNA molecules were synthesized containing predominantly human SRP RNA but possessing M. jannaschii SRP RNA-derived substitutions. Activities of the chimeric RNAs were measured with respect to protein SRP19 and the methionine-rich RNA-binding domain of protein SRP54 (SRP54M). Changing A213 and A214 to a uridine has no effect on the SRP19-dependent binding of SRP54M. Instead, the two base pairs C189-G210 and C190-G209, positioned between the conserved binding site of SRP54 and the asymmetric loop, are critical for conveying SRP19 dependency. Furthermore, the nucleotide composition of five base pairs surrounding the asymmetric loop affects binding of SRP54M significantly. These results demonstrate that subtle, and not easily perceived, structural differences are of crucial importance in the assembly of mammalian SRP.     

A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.     

Saccharomyces SRP RNA secondary structures: a conserved S-domain and extended Alu-domain

The contribution made by the RNA component of signal recognition particle (SRP) to its function in protein targeting is poorly understood. We have generated a complete secondary structure for Saccharomyces cerevisiae SRP RNA, scR1. The structure conforms to that of other eukaryotic SRP RNAs. It is rod-shaped with, at opposite ends, binding sites for proteins required for the SRP functions of signal sequence recognition (S-domain) and translational elongation arrest (Alu-domain). Micrococcal nuclease digestion of purified S. cerevisiae SRP separated the S-domain of the RNA from the Alu-domain as a discrete fragment. The Alu-domain resolved into several stable fragments indicating a compact structure. Comparison of scR1 with SRP RNAs of five yeast species related to S. cerevisiae revealed the S-domain to be the most conserved region of the RNA. Extending data from nuclease digestion with phylogenetic comparison, we built the secondary structure model for scR1. The Alu-domain contains large extensions, including a sequence with hallmarks of an expansion segment. Evolutionarily conserved bases are placed in the Alu- and S-domains as in other SRP RNAs, the exception being an unusual GU(4)A loop closing the helix onto which the signal sequence binding Srp54p assembles (domain IV). Surprisingly, several mutations within the predicted Srp54p binding site failed to disrupt SRP function in vivo. However, the strength of the Srp54p-scR1 and, to a lesser extent, Sec65p-scR1 interaction was decreased in these mutant particles. The availability of a secondary structure for scR1 will facilitate interpretation of data from genetic analysis of the RNA.     

Genetic and biochemical analysis of the fission yeast ribonucleoprotein particle containing a homolog of Srp54p.

Mammalian signal recognition particle (SRP), a complex of six polypeptides and one 7SL RNA molecule, is required for targeting nascent presecretory proteins to the endoplasmic reticulum (ER). Earlier work identified a Schizosaccharomyces pombe homolog of human SRP RNA and showed that it is a component of a particle similar in size and biochemical properties to mammalian SRP. The recent cloning of the gene encoding a fission yeast protein homologous to Srp54p has made possible further characterization of the subunit structure, subcellular distribution, and assembly of fission yeast SRP. S. pombe SRP RNA and Srp54p co-sediment on a sucrose velocity gradient and coimmunoprecipitate, indicating that they reside in the same complex. In vitro assays demonstrate that fission yeast Srp54p binds under stringent conditions to E. coli SRP RNA, which consists essentially of domain IV, but not to the full-length cognate RNA nor to an RNA in which domain III has been deleted in an effort to mirror the structure of bacterial homologs. Moreover, the association of S. pombe Srp54p with SRP RNA in vivo is disrupted by conditional mutations not only in domain IV, which contains its binding site, but in domains I and III, suggesting that the particle may assemble cooperatively. The growth defects conferred by mutations throughout SRP RNA can be suppressed by overexpression of Srp54p, and the degree to which growth is restored correlates inversely with the severity of the reduction in protein binding. Conditional mutations in SRP RNA also reduce its sedimentation with the ribosome/membrane pellet during cell fractionation. Finally, immunoprecipitation under native conditions of an SRP-enriched fraction from [35S]-labeled fission yeast cells suggests that five additional polypeptides are complexed with Srp54p; each of these proteins is similar in size to a constituent of mammalian SRP, implying that the subunit structure of this ribonucleoprotein is conserved over vast evolutionary distances.     

Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET

The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure of Ffh alone and in the complex with 4.5S RNA by measuring distances between different positions within Ffh and between Ffh and 4.5S RNA by fluorescence resonance energy transfer (FRET). According to the FRET distances, NG and M domains in free Ffh are in close contact, as in the A/A arrangement in the crystal structure of Ffh from Thermus aquaticus, in agreement with the formation of a crosslink between cysteine residues at two critical positions in the G and M domains. Upon Ffh binding to 4.5S RNA or a 61 nucleotide fragment comprising internal loops A-C, the G and M domains move apart to assume a more open conformation, as indicated by changes of FRET distances. The movement is smaller when Ffh binds to a 49 nucleotide fragment of 4.5S RNA comprising only internal loops A and B, i.e. lacking the binding site of the NG domain. The FRET results suggest that in the SRP complex 4.5S RNA is present in a bent, rather than extended, conformation. The domain rearrangement of Ffh that takes place upon formation of the SRP is probably important for subsequent steps of membrane targeting, including interactions with the translating ribosome and the SRP receptor.     

Determinants of apical loop-internal loop RNA-RNA interactions involving the HCV IRES

Domain II of the hepatitis C virus internal ribosome entry site is a major RNA structure involved in the viral mRNA translation. It comprises four different structural domains. We performed in vitro selection against the apical loop of the domain II and we identified RNA aptamers folding as an imperfect hairpin with an internal loop of interacting with the apical loop of the domain II. This RNA-RNA interaction creates apical loop-internal loop complex. The aptamer binds the target with an apparent K(d) of 35nM. In this study, the main structural elements of the target and the aptamer involved in the formation of the complex are characterized by mutation, deletion, and RNase probing analysis. We demonstrate that a complementary loop flanked by G,C rich upper and lower stems are crucial for such RNA-RNA interactions.