Ribosomal RNA identity elements for ricin A-chain recognition and catalysis

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose at position A4324 in eukaryotic 28 S rRNA. The requirements for the recognition by ricin A-chain of this nucleotide and for the catalysis of cleavage were examined using a synthetic oligoribonucleotide that reproduces the sequence and the secondary structure of the RNA domain (a helical stem, a bulged nucleotide, and a 17-member single-stranded loop). The wild-type RNA (35mer) and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type oligoribonucleotide the ricin A-chain catalyzed reaction has a Km of 13.55 microM and a Kcat of 0.023 min-1. Recognition and catalysis by ricin A-chain has an absolute requirement for A at the position that corresponds to 4324. The helical stem is also essential; however, the number of base-pairs can be reduced from the seven found in 28 S rRNA to three without loss of identity. The nature of these base-pairs can affect catalysis. A change of the second set from one canonical (G.C) to another (U.A) reduces sensitivity to ricin A-chain; whereas, a change of the third pair (U.A----G.C) produces supersensitivity. The bulged nucleotide does not contribute to identification. Hydrolysis is affected by altering the nucleotides in the universal sequence surrounding A4324 or by changing the position in the loop of the tetranucleotide GA(ricin)GA: all of these mutants have a null phenotype. If ribosomes are treated first with alpha-sarcin to cleave the phosphodiester bond at G4325 ricin can still catalyze depurination at A4324. This implies that cleavage by alpha-sarcin at the center of what has been presumed to be a 17 nucleotide single-stranded loop in 28 S rRNA produces ends that are constrained in some way. On the other hand, hydrolysis by alpha-sarcin of the corresponding position in the synthetic oligoribonucleotide prevents recognition by ricin A-chain. The results suggest that the loop has a complex structure, affected by ribosomal proteins, and this bears on the function in protein synthesis of the alpha-sarcin/ricin rRNA domain.     

The cytotoxins alpha-sarcin and ricin retain their specificity when tested on a synthetic oligoribonucleotide (35-mer) that mimics a region of 28 S ribosomal ribonucleic acid

An oligoribonucleotide (35-mer) that mimics the alpha-sarcin and the ricin region of eukaryotic 28 S rRNA was transcribed in vitro from a synthetic template with T7 RNA polymerase and was used to test whether the specificity of the hydrolysis by the toxins was retained. alpha-Sarcin, at a low concentration, cleaved a single phosphodiester bond on the 3' side of a guanosine residue in the synthetic oligomer that corresponds to G-4325 in 28 S rRNA, the site of action of the toxin in intact ribosomes. At a high concentration of alpha-sarcin, the substrate (35-mer) was hydrolyzed after each of its purines. alpha-Sarcin was without an effect on a synthetic RNA (20-mer) that reproduces the near universal sequence of nucleotides in the loop, but lacks the stem, of the toxin's domain. Thus, the specificity of the attack of alpha-sarcin on a precise region of 28 S rRNA appears to be contingent on the sequence of the nucleotides and the structure of the domain. Ricin depurinated a nucleotide in the synthetic oligomer (35-mer), and in the presence of aniline the phosphoribose backbone was cleaved at a position that conforms to A-4324 in 28 S rRNA, the site of action of the toxin in vivo.     

Ribosomal RNA identity elements for ricin A-chain recognition and catalysis. Analysis with tetraloop mutants.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose of the adenosine at position 4324 in eukaryotic 28 S rRNA. Ricin A-chain will also catalyze depurination in naked prokaryotic 16 S rRNA; the adenosine is at position 1014 in a GAGA tetraloop. The rRNA identity elements for recognition by ricin A-chain and for the catalysis of cleavage were examined using synthetic GAGA tetraloop oligoribonucleotides. The RNA designated wild-type, an oligoribonucleotide (19-mer) that approximates the structure of the ricin-sensitive site in 16 S rRNA, and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type tetraloop oligoribonucleotide the ricin A-chain-catalyzed reaction has a Km of 5.7 microM and a Kcat of 0.01 min-1. The toxin alpha-sarcin, which cleaves the phosphodiester bond on the 3' side of G4325 in 28 S rRNA, does not recognize the tetraloop RNA, although alpha-sarcin does affect a larger synthetic oligoribonucleotide that has a 17-nucleotide loop with a GAGA sequence; thus, there is a clear divergence in the identity elements for the two toxins. Mutants were constructed with all of the possible transitions and transversions of each nucleotide in the GAGA tetraloop; none was recognized by ricin A-chain. Thus, there is an absolute requirement for the integrity of the GAGA sequence in the tetraloop. The helical stem of the tetraloop oligoribonucleotide can be reduced to three base-pairs, indeed, to two base-pairs if the temperature is decreased, without affecting recognition; the nature of these base-pairs does not influence recognition or catalysis by ricin A-chain. If the tetraloop is opened so as to form a GAGA-containing hexaloop, recognition by ricin A-chain is lost. This suggests that during the elongation cycle, a GAGA tetraloop either exists or is formed in the putative 17-member single-stranded region of the ricin domain in 28 S rRNA and this bears on the mechanism of protein synthesis.     

The ribosomal RNA identity elements for ricin and for alpha-sarcin: mutations in the putative CG pair that closes a GAGA tetraloop.

alpha-Sarcin is a ribonuclease that cleaves the phosphodiester bond on the 3' side of G4325 in 28S rRNA; ricin A-chain is a RNA N-glycosidase that depurinates the 5' adjacent A4324. These single covalent modifications inactivate the ribosome. An oligoribonucleotide that reproduces the structure of the sarcin/ricin domain in 28S rRNA was synthesized and mutations were constructed in the 5' C and the 3' G that surround a GAGA tetrad that has the sites of toxin action. Covalent modification of the RNA by ricin, but not by alpha-sarcin, requires a Watson-Crick pair to shut off a putative GAGA tetraloop. Either the recognition elements for the two toxins are different despite their catalyzing covalent modification of adjacent nucleotides in 28S rRNA or there are transitions in the conformation of the alpha-sarcin/ricin domain in 28S rRNA and one conformer is recognized by alpha-sarcin and the other by ricin A-chain.     

Mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes

We have studied on the mechanism of ricin action on rat liver ribosomes and present evidence which shows that the toxin inactivates ribosomes by modifying two bases at positions G-4323 and A-4324 of 28S rRNA adjacent to alpha-sarcin cleavage site. Further results showing that those phosphodiester bonds are very labile against alkaline digestion and aniline-treatment strongly suggest that these purine bases are removed by N-glycosidase activity of the toxin. In parallel, we also present evidence showing that abrin and modeccin have the same activity on eukaryotic ribosomes as ricin does.     

The action mode of the ribosome-inactivating protein α-sarcin

Based on the tertiary structure of the ribosome-inactivating protein alpha-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected. In this study, we found that the head-to-tail interaction between the first amino beta-strand and the last carboxyl beta-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated beta-sheet is indispensable for catalyzing both substrates, suggesting that alpha-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino beta-hairpin and that of the loop L3 in alpha-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the beta-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the beta-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes.     

High-resolution NMR study of a synthetic oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate for the cytotoxic protein, ricin.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Its substrate domain forms a double helical stem and a 17-base loop that includes the sequence GAGA, the second adenosine of which corresponds to A4324. Recently, studies of mutant RNAs have shown that the four-nucleotide loop, GAGA, can function as a substrate for ricin. To investigate the structure that is recognized by ricin, we studied the properties of a short synthetic substrate, the dodecaribonucleotide r-CUCAGAGAUGAG, which forms a RNA hairpin structure with a GABA loop and a stem of four base pairs. The results of NMR spectroscopy allowed us to construct the solution structure of this oligonucleotide by restrained molecular-dynamic calculations. We found that the stem region exists as an A-form duplex. 5G and 8A in the loop region form an unusual G:A base pair, and the phosphodiester backbone has a turn between 5G and 6A. This turn seems to help ricin to gain access to 6A which is the only site of depurination in the entire structure. The overall structure of the GAGA loop is similar to those of the GAAA and GCAA loops that have been described but that are not recognized by ricin. Therefore, in addition to the adenosine at the depurination site, the neighboring guanosine on the 3' side (7G) may also play a role in the recognition mechanism together with 5G and 8A.     

The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

Ricin is a potent cytotoxic protein derived from the higher plant Ricinus communis that inactivates eukaryotic ribosomes. In this paper we have studied the mechanism of action of ricin A-chain on rat liver ribosomes in vitro. Our findings indicate that the toxin inactivates the ribosomes by modifying both or either of two nucleoside residues, G4323 and A4324, in 28 S rRNA. These nucleotides are located close to the alpha-sarcin cleavage site and become resistant to all ribonucleases tested. The examination of the lability of phosphodiester bonds of these nucleotides to both mild alkaline digestion and aniline treatment at acidic pH suggests that the base of A4324 is removed by the toxin. This unique activity of ricin A-chain was also observed when naked 28 S rRNA is used as a substrate, indicating that the toxin directly acts on the RNA. Similar activity on 28 S rRNA is also exhibited by abrin and modeccin, ricin-related toxins, suggesting a general mechanistic pathway for ribosome inactivation by lectin toxins.     

A novel ribotoxin with ribonuclease activity that specifically cleaves a single phosphodiester bond in rat 28S ribosomal RNA and inactivates ribosome

A unique ribonuclease named Biota orientalis ribonuclease (Biota orientalis RNase) is purified to homogeneity from mature seeds of oriental arborvitae (Biota orientalis). The molecular mass of Biota orientalis RNase is about 13 kDa. When the concentration of Mg(2+) is 25 mM in the incubation buffer, the ribonuclease specifically cleaves the phosphodiester bond between C4453 and A4454 in region K (a region in domain VII) of 28S RNA in rat ribosome, resulting in inactivation of ribosome. Thus, it is a ribotoxin similar to alpha-sarcin. The region around C4453-A4454 in rat 28S rRNA is named "Biota orientalis RNase region." Rat ribosome treated by Biota orientalis RNase produces a small RNA fragment (S-fragment) that contains 333 nucleotides from the 3'-terminus of rat 28S rRNA. The distance between the cleavage-sites of alpha-sarcin (G4325) and Biota orientalis RNase (C4453) is 128 nucleotides. Under restricted conditions (25 mM Mg(2+)), the substrate specificity of Biota orientalis RNase is extremely high: it acts only on the "Biota orientalis RNase region" of the largest RNA in ribosomes from certain eukaryotes. The ribosome specifically damaged by Biota orientalis RNase is unable to EF-1alpha-dependently bind aminoacyl-tRNA, whereas the formation of the EF-2/GDP/ribosome complex is not affected. It is proposed that Biota orientalis RNase inactivates ribosome at least partially by interfering with the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosome. Biota orientalis RNase might be a useful tool in studying the structure/function of ribosome.     

The mechanism of action of the cytotoxic lectin from Phoradendron californicum: the RNA N-glycosidase activity of the protein

A toxic lectin from Phoradendron californicum (PCL) was found to inactivate catalytically 60 S ribosomal subunits of rabbit reticulocytes, resulting in the inhibition of protein synthesis. To study the mechanism of action of PCL, rat liver ribosomes were treated with the toxin and the extracted rRNA was treated with aniline. A fragment containing about 450 nucleotides was released from the 28 S rRNA. Analysis of the nucleotide sequence of the fragment revealed that the aniline-sensitive phosphodiester bond was between A4324 and G4325 of the 28 S rRNA. These results indicate that PCL inactivates the ribosomes by cleaving an N-glycosidic bond at A4324 of 28 S rRNA in the ribosomes as does ricin A-chain.     

Microinjected oligonucleotides complementary to the alpha-sarcin loop of 28 S RNA abolish protein synthesis in Xenopus oocytes

The integrity of the alpha-sarcin loop in 28 S ribosomal RNA is critical during protein synthesis. The toxins alpha-sarcin, ricin, Shiga toxin, and Shiga-like toxin inhibit protein synthesis in oocytes by attacking specific nucleotides within this loop (Ackerman, E.J., Saxena, S. K., and Ulbrich, N. (1988) J. Biol. Chem. 263, 17076-17083; Saxena, S.K., O'Brien, A.D., and Ackerman, E.J. (1989) J. Biol. Chem. 264, 596-601). We injected Xenopus oocytes with deoxyoligonucleotides complementary to the 17-nucleotide alpha-sarcin loop of Xenopus 28 S rRNA. Only injected oligonucleotides fully covering the alpha-sarcin loop or slightly beyond inhibited oocyte protein synthesis. Shorter alpha-sarcin domain deoxyoligonucleotides complementary to the alpha-sarcin and ricin sites but not spanning the entire loop were less effective inhibitors of protein synthesis. The alpha-sarcin domain oligonucleotides covering the entire loop were more effective inhibitors of protein synthesis than injected cycloheximide at equivalent concentrations. Control oligonucleotides complementary to nine other regions of Xenopus 28 S rRNA as well as universal M13 DNA sequencing primers had no effect on oocyte protein synthesis. Oligonucleotides complementary to the highly conserved alpha-sarcin domain therefore represent an alternative to catalytic toxins for causing cell death and may prove effective in immunotherapy.     

Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

A potential RNA drug target in the hepatitis C virus internal ribosomal entry site

Subdomain IlId from the hepatitis C virus (HCV) internal ribosome entry site (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequence to IlId, to explore the structural features of this subdomain. The proposed secondary structure of IlId is comprised of two 3 bp helical regions separated by an internal loop and closed at one end by a 6-nt terminal loop. NMR and molecular modeling were used interactively to formulate a validated model of the three-dimensional structure of IlId. We found that this fragment contains several noncanonical structural motifs and non-Watson-Crick base pairs, some of which are common to other RNAs. In particular, a motif characteristic of the rRNA alpha-sarcin/ricin loop was located in the internal loop. The terminal loop, 5'-UUGGGU, was found to fold to form a trinucleotide loop closed by a trans-wobble U.G base pair. The sixth nucleotide was bulged out to allow stacking of this U.G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirmed the importance of each structural motif within IIId for IRES function. These findings may provide clues as to host cellular proteins that play a role in IRES-directed translation and, in particular, the mechanism through which host ribosomes are sequestered for viral function.     

The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity

alpha-Sarcin selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA, that inactivates the ribosome. The elucidation of the three-dimensional solution structure of this 150 residue enzyme is a crucial step towards understanding alpha-sarcin's conformational stability, ribonucleolytic activity, and its exceptionally high level of specificity. Here, the solution structure has been determined on the basis of 2658 conformationally relevant distances restraints (including stereoespecific assignments) and 119 torsional angular restraints, by nuclear magnetic resonance spectroscopy methods. A total of 60 converged structures have been computed using the program DYANA. The 47 best DYANA structures, following restrained energy minimization by GROMOS, represent the solution structure of alpha-sarcin. The resulting average pairwise root-mean-square-deviation is 0.86 A for backbone atoms and 1.47 A for all heavy atoms. When the more variable regions are excluded from the analysis, the pairwise root-mean-square deviation drops to 0.50 A and 1.00 A, for backbone and heavy atoms, respectively. The alpha-sarcin structure is similar to that reported for restrictocin, although some differences are clearly evident, especially in the loop regions. The average rmsd between the structurally aligned backbones of the 47 final alpha-sarcin structures and the crystal structure of restrictocin is 1.46 A. On the basis of a docking model constructed with alpha-sarcin solution structure and the crystal structure of a 29-nt RNA containing the sarcin/ricin domain, the regions in the protein that could interact specifically with the substrate have been identified. The structural elements that account for the specificity of RNA recognition are located in two separate regions of the protein. One is composed by residues 51 to 55 and loop 5, and the other region, located more than 11 A away in the structure, is the positively charged segment formed by residues 110 to 114.     

Effects of antisense DNA against the alpha-sarcin stem-loop structure of the ribosomal 23S rRNA.

Antisense DNAs complementary against various sequences of the alpha-sarcin domain (C2646-G2674) of 23S rRNA from Escherichia coli were hybridized to naked 23S rRNA as well as to 70S ribosomes. Saturation levels of up to 0.4 per 70S ribosome were found, the identical fraction was susceptible to the attack of the RNase alpha-sarcin. The hybridization was specific as demonstrated with RNase H digestion, sequencing the resulting fragments and blockage of the action of alpha-sarcin. The RNase alpha-sarcin seems to approach its cleavage site from the 3' half of the loop of the alpha-sarcin domain. Hybridization is efficiently achieved at 37 degrees C and can extend at least into the 3' strand of the stem of the alpha-sarcin domain. However, the inhibition of alpha-sarcin activity is observed at 30 degrees C but not at 37 degrees C. For a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25 degrees C. The results imply that the alpha-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the alpha-sarcin domain.     

Ribozymes correctly cleave a model substrate and endogenous RNA in vivo

The alpha-sarcin domain of 28 S RNA in Xenopus oocytes is attacked by several catalytic toxins (e.g. alpha-sarcin and ricin) that abolish protein synthesis. We synthesized 6 ribozymes targeted to the alpha-sarcin domain and to an oligoribonucleotide (34-mer) that mimics this domain. Sarcin ribozyme 5 (SR5) efficiently cleaved after the CUC site in the synthetic 34-mer in vitro at 50 degrees C. SR5 also cut the same site when both substrate and ribozyme were coinjected or injected separately into oocytes at 18 degrees C. Correct cleavage in vivo was shown by isolating and sequencing the large cleavage fragment. The cleavage reaction appeared to function equally well in the oocyte nucleus and cytoplasm. SR5 also correctly cleaved endogenous 28 S RNA in oocytes, although cutting was much less efficient than with alpha-sarcin. We therefore demonstrated that a ribozyme specifically cuts both a model substrate and a cellular RNA in vivo. Earlier work showed that certain injected deoxyoligonucleotides complementary to the alpha-sarcin region abolish protein synthesis. Oocyte protein synthesis was also abolished by an SR5 containing a single G----U substitution that inactivates RNA catalysis, indicating that SR5's translational suppression was perhaps due to antisense function rather than ribozyme cleavage.     

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.     

Investigating the structural basis of purine specificity in the structures of MS2 coat protein RNA translational operator hairpins

We have determined the structures of complexes between the phage MS2 coat protein and variants of the replicase translational operator in order to explore the sequence specificity of the RNA–protein interaction. The 19-nt RNA hairpins studied have substitutions at two positions that have been shown to be important for specific binding. At one of these positions, –10, which is a bulged adenosine (A) in the stem of the wild-type operator hairpin, substitutions were made with guanosine (G), cytidine (C) and two non-native bases, 2-aminopurine (2AP) and inosine (I). At the other position, –7 in the hairpin loop, the native adenine was substituted with a cytidine. Of these, only the G-10, C-10 and C-7 variants showed interpretable density for the RNA hairpin. In spite of large differences in binding affinities, the structures of the variant complexes are very similar to the wild-type operator complex. For G-10 substitutions in hairpin variants that can form bulges at alternative places in the stem, the binding affinity is low and a partly disordered conformation is seen in the electron density maps. The affinity is similar to that of wild-type when the base pairs adjacent to the bulged nucleotide are selected to avoid alternative conformations. Both purines bind in a very similar way in a pocket in the protein. In the C-10 variant, which has very low affinity, the cytidine is partly inserted in the protein pocket rather than intercalated in the RNA stem. Substitution of the wild-type adenosine at position –7 by pyrimidines gives strongly reduced affinities, but the structure of the C-7 complex shows that the base occupies the same position as the A-7 in the wild-type RNA. It is stacked in the RNA and makes no direct contact with 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.