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.     

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.     

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.     

Important role of the tetraloop region of 4.5S RNA in SRP binding to its receptor FtsY

Binding of Escherichia coli signal recognition particle (SRP) to its receptor, FtsY, requires the presence of 4.5S RNA, although FtsY alone does not interact with 4.5S RNA. In this study, we report that the exchange of the GGAA tetraloop sequence in domain IV of 4.5S RNA for UUCG abolishes SRP-FtsY interaction, as determined by gel retardation and membrane targeting experiments, whereas replacements with other GNRA-type tetraloops have no effect. A number of other base exchanges in the tetraloop sequence have minor or intermediate inhibitory effects. Base pair disruptions in the stem adjacent to the tetraloop or replacement of the closing C-G base pair with G-C partially restored function of the otherwise inactive UUCG mutant. Chemical probing by hydroxyl radical cleavage of 4.5S RNA variants show that replacing GGAA with UUCG in the tetraloop sequence leads to structural changes both within the tetraloop and in the adjacent stem; the latter change is reversed upon reverting the C-G closing base pair to G-C. These results show that the SRP-FtsY interaction is strongly influenced by the structure of the tetraloop region of SRP RNA, in particular the tetraloop stem, and suggest that both SRP RNA and Ffh undergo mutual structural adaptation to form SRP that is functional in the interaction with the receptor, FtsY.     

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.     

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.     

Conformity of RNAs that interact with tetranucleotide loop binding proteins.

A group of RNA binding proteins, termed tetraloop binding proteins, includes ribosomal protein S15 and protein SRP19 of signal recognition particle. They are primary RNA binding proteins, recognize RNA tetranucleotide loops with a GNAR consensus motif, and require a helical region located adjacent to the tetraloop. Closely related RNA structures that fit these criteria appear in helix 6 of SRP RNA, in helices 22 and 23A of 16 S ribosomal RNA, and, as a pseudoknot, in the regulatory region of the rpsO gene.     

Solution structure of a SRP19 binding domain in human SRP RNA

Assembly of the human signal recognition particle (SRP) requires SRP19 protein to bind to helices 6 and 8 of SRP RNA. In the present study, structure of a 29-mer RNA composing the SRP19 binding site in helix 6 was determined by NMR spectroscopy. The two A:C mismatches were continuously stacked to each other and formed wobble type A:C base pairs. The GGAG tetraloop in helix 6 was found to adopt a similar conformation to that of GNRA tetraloop, suggesting that these tetraloops are included in an extensive new motif GNRR. Compared with the crystal structure of helix 6 in complex with SRP19 determined previously, the GGAG tetraloop in the complex was found to adopt a similar conformation to the free form, although the loop structure becomes more open upon SRP19 binding. Thus, SRP19 is thought to recognize the overall fold of the GGAG loop.     

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP-receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP-receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP-receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo.     

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.     

The 54-kD protein of signal recognition particle contains a methionine- rich RNA binding domain

Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V. R. Lingappa. 1986. Annu. Rev. Cell Biol. 2:499- 516). It consists of SRP7S RNA and six proteins. The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides. The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle. We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle. Deletion of the 21 COOH- terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA. Further deletions abolish SRP19 binding to SRP7S RNA. The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro. Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay. The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA. We suggest that the methionine-rich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.     

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.     

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.     

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.     

The Alu domain homolog of the yeast signal recognition particle consists of an Srp14p homodimer and a yeast-specific RNA structure

The mammalian Alu domain of the signal recognition particle (SRP) consists of a heterodimeric protein SRP9/14 and the Alu portion of 7SL RNA and comprises the elongation arrest function of the particle. To define the domain in Saccharomyces cerevisiae SRP that is homologous to the mammalian Alu domain [Alu domain homolog in yeast (Adhy)], we examined the assembly of a yeast protein homologous to mammalian SRP14 (Srp14p) and scR1 RNA. Srp14p binds as a homodimeric complex to the 5' sequences of scR1 RNA. Its minimal binding site consists of 99 nt. (Adhy RNA), comprising a short hairpin structure followed by an extended stem. As in mammalian SRP9/14, the motif UGUAAU present in most SRP RNAs is part of the Srp14p binding sites as shown by footprint and mutagenesis studies. In addition, certain basic amino acid residues conserved between mammalian SRP14 and Srp14p are essential for RNA binding in both proteins. These findings confirm the common ancestry of the yeast and the mammalian components and indicate that Srp14p together with Adhy RNA represents the Alu domain homolog in yeast SRP that may comprise its elongation arrest function. Despite the similarities, Srp14p selectively recognizes only scR1 RNA, revealing substantial changes in RNA-protein recognition as well as in the overall structure of the complex. The alignment of the three yeast SRP RNAs known to date suggests a common structure for the putative elongation arrest domain of all three organisms.     

Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA.

The interaction of protein SRP54M from the human signal recognition particle with SRP RNA was studied by systematic site-directed mutagenesis of the RNA molecule. Protein binding sites were identified by the analysis of mutations that removed individual SRP RNA helices or disrupted helical sections in the large SRP domain. The strongest effects on the binding activity of a purified polypeptide that corresponds to the methionine-rich domain of SRP54 (SRP54M) were caused by changes in helix 8 of the SRP RNA. Binding of protein SRP19 was diminished significantly by mutations in helix 6 and was stringently required for SRP54M to associate. Unexpectedly, mutant RNA molecules that resembled bacterial SRP RNAs were incapable of interaction with SRP54M, showing that protein SRP19 has an essential and direct role in the formation of the ternary complex with SRP54 and SRP RNA. Our findings provide an example for how, in eukaryotes, an RNA function has become protein dependent.     

Protein-induced conformational changes of RNA during the assembly of human signal recognition particle

The human signal recognition particle (SRP) is a large RNA-protein complex that targets secretory and membrane proteins to the endoplasmic reticulum membrane. The S domain of SRP is composed of roughly half of the 7SL RNA and four proteins (SRP19, SRP54, and the SRP68/72 heterodimer). In order to understand how the binding of proteins induces conformational changes of RNA and affects subsequent binding of other protein subunits, we have performed chemical and enzymatic probing of all S domain assembly intermediates. Ethylation interference experiments show that phosphate groups in helices 5, 6 and 7 that are essential for the binding of SRP68/72 are all on the same face of the RNA. Hydroxyl radical footprinting and dimethylsulphate (DMS) modifications show that SRP68/72 brings the lower part of helices 6 and 8 closer. SRP68/72 binding also protects the SRP54 binding site (helix 8 asymmetric loop) from chemical modification and RNase cleavage, whereas, in the presence of both SRP19 and SRP68/72, the long strand of helix 8 asymmetric loop becomes readily accessible to chemical and enzymatic probes. These results indicate that the RNA platform observed in the crystal structure of the SRP19-SRP54M-RNA complex already exists in the presence of SRP68/72 and SRP19. Therefore, SRP68/72, together with SRP19, rearranges the 7SL RNA in an SRP54 binding competent state.     

Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III

Members of the RNase III family of double-stranded RNA (dsRNA) endonucleases are important enzymes of RNA metabolism in eukaryotic cells. Rnt1p is the only known member of the RNase III family of endonucleases in Saccharomyces cerevisiae. Previous studies have shown that Rnt1p cleaves dsRNA capped by a conserved AGNN tetraloop motif, which is a major determinant for Rnt1p binding and cleavage. The solution structure of the dsRNA-binding domain (dsRBD) of Rnt1p bound to a cognate RNA substrate revealed the structural basis for binding of the conserved tetraloop motif by alpha-helix 1 of the dsRBD. In this study, we have analyzed extensively the effects of mutations of helix 1 residues that contact the RNA. We show, using microarray analysis, that mutations of these amino acids induce substrate-specific processing defects in vivo. Cleavage kinetics and binding studies show that these mutations affect RNA cleavage and binding in vitro to different extents and suggest a function for some specific amino acids of the dsRBD in the catalytic positioning of the enzyme. Moreover, we show that 2'-hydroxyl groups of nucleotides of the tetraloop or adjacent base pairs predicted to interact with residues of alpha-helix 1 are important for Rnt1p cleavage in vitro. This study underscores the importance of a few amino acid contacts for positioning of a dsRBD onto its RNA target, and implicates the specific orientation of helix 1 on the RNA for proper positioning of the catalytic domain.     

Complexes with truncated RNAs from the large domain of Archaeoglobus fulgidus signal recognition particle

Protein SRP19 is an important component of the signal recognition particle (SRP) as it promotes assembly of protein SRP54 with SRP RNA and recognizes a tetranucleotide loop. Structural features and RNA binding activities of SRP19 of the hyperthermophilic archaeon Archaeoglobus fulgidus were investigated. An updated alignment of SRP19 sequences predicted three conserved regions and two alpha-helices. With Af-SRP RNA the Af-SRP54 protein assembled into an A. fulgidus SRP which remained intact for many hours. Stable complexes were formed between Af-SRP19 and truncated SRP RNAs, including a 36-residue fragment representing helix 6 of A. fulgidus SRP RNA.     

Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites.

Signal recognition particle (SRP), a ribonucleoprotein composed of six polypeptides and one RNA subunit, serves as an adaptor between the cytoplasmic protein synthetic machinery and the translocation apparatus of the endoplasmic reticulum. To begin constructing a functional map of the 7SL RNA component of SRP, we extensively mutagenized the Schizosaccharomyces pombe SRP7 gene. Phenotypes are reported for fifty-two mutant alleles derived from random point mutagenesis, seven alleles created by site-directed mutagenesis to introduce restriction sites into the SRP7 gene, nine alleles designed to pinpoint conditional lesions, and three alleles with extra nucleotides inserted at position 84. Our data indicate that virtually all single nucleotide changes as well as many multiple substitutions in this highly structured RNA are phenotypically silent. Six lethal alleles and eleven which result in sensitivity to the combination of high temperature and elevated osmotic strength were identified. These mutations cluster in conserved regions which, in the mammalian RNA, are protected from nucleolytic agents by SRP proteins. The effects of mutations in the presumptive binding site for a fission yeast SRP 9/14 homolog indicate that both the identity of a conserved residue and the secondary structure within which it is embedded are functionally important. The phenotypes of mutations in Domain IV suggest particular residues as base-specific contacts for the fission yeast SRP54 protein. A single allele which confers temperature-sensitivity in the absence of osmotic perturbants was identified in this study; the growth properties of the mutant strain suggest that the encoded RNA is somewhat defective even at the permissive temperature, and is most likely unable to correctly assemble with SRP proteins at the nonpermissive temperature.