Demonstration of RNA N-glycosidase activity of a Vero toxin (VT2 variant) produced by Escherichia coli O91:H21 from a patient with the hemolytic uremic syndrome

A new Vero toxin purified from Escherichia coli O91:H21 isolated from a patient with the hemolytic uremic syndrome (VT2vh) was shown to inhibit elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes, resulting in inhibition of protein synthesis in rabbit reticulocytes. VT2vh, like Shiga toxin, VT1 and VT2, showed RNA N-glycosidase activity and cleaved the N-glycosidic bond of the adenosine residue at position 4324 in 28S ribosomal RNA.     

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.     

Cross-protection against challenge by intravenous Escherichia coli verocytotoxin 1 (VT1) in rabbits immunized with VT2 toxoid

Rabbits challenged intravenously with Escherichia coli verocytotoxin (VT1, Shiga toxin 1, Stx1) die after developing diarrhea and paralysis, and this outcome can be prevented by pre-immunization with VT1 toxoid. In nonimmune rabbits, intravenously administered 125I-VT1 binds to the central nervous system and gastrointestinal tract, whereas in immunized animals, these organs are spared and the toxin localizes in the liver and spleen. In rabbits immunized with either VT1 or VT2 toxoids, both the homologous or heterologous toxins are prevented from binding to target organs. This has lead to the advancement of a hypothesis that cross-protection in vivo can be induced to both toxins by immunization with a toxoid even though these toxins do not exhibit cross-neutralization in vitro. It was shown that rabbits immunized with VT2 were fully protected from the intravenous administration of 10 LD50 and 50 LD50 of VT1, and this correlated directly with the protection from binding of this toxin to target organs. These findings have important implications on the design of the vaccination strategies to prevent human VT-mediated diseases and also validate the concept of testing for immunity to VT by monitoring the inhibition of binding of the 125I-VT to target organs in preference to performing LD50 assays.     

Shiga toxin 1 is more dependent on the P proteins of the ribosomal stalk for depurination activity than Shiga toxin 2

Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated ribosomes less in a strain lacking P1/P2 on the ribosome and in the cytosol (ΔP2) than in a strain lacking P1/P2 on the ribosome, but containing free P2 in the cytosol (ΔP1). To determine if cytoplasmic P proteins facilitated depurination, Stx1A and Stx2A were expressed in the P0ΔAB mutant, in which the binding sites for P1/P2 were deleted on the ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated ribosomes less in P0ΔAB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated ribosomes in P0ΔAB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ΔP1, but not P0ΔAB ribosomes increased upon addition of purified P1α/P2βin vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate depurination by Stx1 by facilitating the access of the toxin to the ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate ribosomes with an incomplete stalk better than Stx1.     

Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1

The binding of Shiga toxin isolated from the bacterium Shigella dysenteriae type 1 to a series of glycolipids and to cells or cell homogenates has been studied. Bound toxin was detected using either 125I-labeled toxin or specific monoclonal antibody and 125I-labeled anti-antibody. Overlay of toxin on thin-layer chromatograms with separated glycolipids and binding to glycolipids coated in microtiter wells established that the toxin specifically bound to Gal alpha 1-4Gal beta (galabiose) placed terminally or internally in the oligosaccharide chain. No glycolipid shown to lack this sequence binds the toxin. Most of the glycolipids with internally placed galabiose were not active, indicating a sterical hindrance for toxin access to the binding epitope. Binding of toxin to HeLa cells in monolayers could be inhibited by preincubation of the toxin with galabiose covalently linked to bovine serum albumin (BSA), but not with free oligosaccharides containing galabiose or with lactose coupled to BSA. This demonstrated that the inhibition is specifically dependent on galabiose and requires multivalency of the disaccharide to be efficient. The inhibitory effect was successively enhanced by increasing the substitution on BSA (7, 18, and 25 mol of galabiose/mol of BSA). The BSA-coupled galabiose could also prevent the cytotoxic effect on HeLa cells (detachment of killed cells). There are cell lines with a dense number of receptor sites, but which are resistant to toxin action (uptake and inhibition of protein synthesis) which may suggest two types of receptor substances which are functionally different and unevenly expressed. In analogy with the mechanism earlier formulated for cholera toxin, we propose glycolipid-bound, bilayer-close galabiose as the functional receptor for membrane penetration of the toxin, while galabiose bound in glycoproteins affords binding sites but is not able to mediate penetration.     

Effects of ricin on the ribosomal sites involved in the interaction of the elongation factors.

The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell-free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)-directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible. Ricin specifically inhibits elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes but has no effect on the non-enzymic binding of aminoacyl-tRNA. Ricin also inhibits formation of the complex elongation-factor-2 - ribosome - nucleotide with GTP, GDP or GMP-P(CH2)P. However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.     

Importance of glycolipid synthesis for butyric acid-induced sensitization to shiga toxin and intracellular sorting of toxin in A431 cells.

The human epidermoid carcinoma cell line A431 becomes highly sensitive to Shiga toxin upon treatment with butyric acid. This strong sensitization (> 1000-fold) is accompanied by an increase in the fraction of cell-associated toxin transported to the Golgi apparatus and to the endoplasmic reticulum (ER). Furthermore, our previous work showed that the length of the fatty acyl chain of Gb3, the Shiga toxin receptor, also was changed (longer fatty acids). We have not investigated the importance of this change by testing whether glycolipid synthesis is required for the changed intracellular sorting and the toxin sensitivity. We demonstrate here that inhibition of glycosphingolipid synthesis by inhibition of N-acyltransferase with fumonisin B1, by inhibition of glucosylceramide synthetase by PDMP or PPMP, or by inhibition of serine palmitoyl transferase by beta-fluoroalanine, inhibited the butyric acid-induced change in sensitivity and the increase in the fraction of cell-associated Shiga toxin transported to the Golgi apparatus and the ER. The block in butyric acid-induced sensitization caused by beta-fluoroalanine could be abolished by simultaneous addition of sphinganine or sphingosine. Thus, the data suggest that the fatty acyl chain length of glycosphingolipids is important for intracellular sorting and translocation of Shiga toxin to the cytosol.     

Inhibition of polypeptide synthesis by tRNA fractions in rat liver cell-free systems.

The mechanism of inhibition of polypeptide synthesis by the addition of a tRNA fraction in a rat liver cell-free system was studied. The inhibition was found to occur at the step of aminoacyl-tRNA binding to ribosomes, in which aminoacyl-tRNA's were mainly responsible for the inhibition. The addition of EF-1 decreased the inhibition by the tRNA fraction. The tRNA fraction inhibited polypeptide synthesis in a polysome-S100 system under conditions in which poly U- and poly A-dependent polypeptide syntheses were not inhibited. The possibility that the aminoacyl-tRNA inhibitory activity functions through improper binding to the ribosomes in the polysome-S100 system is discussed.     

Characterization of purified Shiga toxin from Shigella dysenteriae 1

Shiga toxin was purified from the culture supernatant of Shigella dysenteriae 1 by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatography. About 1.6 mg of purified Shiga toxin was obtained from 15 liters of culture with a yield of about 27%. The molecular weight of purified Shiga toxin was estimated to be 62,000. The toxin consisted of A and B subunits with molecular weights of about 30,000 and 5,000-6,000, respectively. The isoelectric point of purified Shiga toxin was 7.0. Purified Shiga toxin showed the following biological activities: lethal toxicity to mice when injected intraperitoneally with an LD50 of 28 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 1 pg and all of the cells at 10 pg; and fluid accumulation in rabbit ileal loops at a concentration of more than 1 microgram.     

Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis.

The B subunit of Shiga toxin and the Shiga-like toxins (SLTs) mediates receptor binding, cytotoxic specificity, and extracellular localization of the holotoxin. While the functional receptor for Shiga toxin, SLT type I (SLT-I), and SLT-II is the glycolipid designated Gb3, SLT-II variant (SLT-IIv) may use a different glycolipid receptor. To identify the domains responsible for receptor binding, localization in Escherichia coli, and recognition by neutralizing monoclonal antibodies, oligonucleotide-directed site-specific mutagenesis was used to alter amino acid residues in the B subunits of Shiga toxin and SLT-IIv. Mutagenesis of a well-conserved hydrophilic region near the amino terminus of the Shiga toxin B subunit rendered the molecule nontoxic but did not affect immunoreactivity or holotoxin assembly. In addition, elimination of one cysteine residue, as well as truncation of the B polypeptide by 5 amino acids, caused a total loss of activity. Changing a glutamate to a glutamine at the carboxyl terminus of the Shiga toxin B subunit resulted in the loss of receptor binding and immunoreactivity. However, the corresponding mutation in the SLT-IIv B subunit (glutamine to glutamate) did not reduce the levels of cytotoxicity but did affect extracellular localization of the holotoxin in E. coli.     

4-Aminopyrazolo[3,4-d]pyrimidine (4-APP) as a novel inhibitor of the RNA and DNA depurination induced by Shiga toxin 1

Shiga toxin 1 (Stx1) catalyses the removal of a unique and specific adenine from 28S RNA in ribosomes (RNA-N-glycosidase activity) and the release of multiple adenines from DNA (DNA glycosylase activity). Added adenine behaves as an uncompetitive inhibitor of the RNA-N-glycosidase reaction binding more tightly to the Stx1–ribosome complex than to the free enzyme. Several purine derivatives and analogues have now been assayed as inhibitors of Stx1. Most of the compounds showed only minor differences in the rank order of activity on the two enzymatic reactions catalysed by Stx1. The survey highlights the importance of the amino group in the 6-position of the pyrimidine ring of adenine. Shifting (2-aminopurine) or substituting (hypoxanthine, 6-mercaptopurine, 6-methylpurine) the group greatly decreases the inhibitory power. The presence of a second ring, besides the pyrimidine one, is strictly required. Substitution, by introducing an additional nitrogen, of the imidazole ring of adenine with triazole leads to loss of inhibitory power, while rearrangement of the nitrogen atoms of the ring from the imidazole to the pyrazole configuration greatly enhances the inhibitory power. Thus 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), the isomer of adenine with the five-membered ring in the pyrazole configuration, is by far the most potent inhibitor of both enzymatic reactions catalysed by Stx1. This finding opens perspectives on therapeutic strategies to protect endothelial renal cells once endocytosis of Stx1 has occurred (haemolytic uraemic syndrome). In the RNA-N-glycosidase reaction 4-APP binds, as adenine, predominantly to the Stx1–ribosome complex (uncompetitive inhibition), while inhibition of the DNA glycosylase activity by both inhibitors is of the mixed type.     

Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1

Evidence is presented that endocytosis is involved in the transport to the cytosol of the cytotoxin from Shigella dysenteriae 1, Shiga toxin, which acts by removal of a single adenine residue in 28-S ribosomal RNA. Inhibition of endocytosis by ATP depletion of the cells prevented toxin uptake. Exposure of HeLa S3 and Vero cells to toxin at low extracellular pH, where translocation to the cytosol, but not endocytosis is inhibited, allowed the toxin to accumulate in a compartment where it was protected against antibodies to the toxin. Upon transfer of the cells to normal medium endocytosed toxin entered the cytosol. Electron microscopical studies of cells exposed at 0 degrees C to a toxin-horseradish peroxidase (HRP) conjugate, or to unconjugated toxin followed by horse antitoxin antibodies and then protein G-gold, revealed that the Shiga toxin binding sites were randomly distributed on the cell surface, without any preference to, for example, coated pits. In contrast, when cells were exposed to toxin at 37 degrees C, the binding sites were preferentially localized in coated pits. The Shiga-HRP conjugate was also seen in endosomes, lysosomes, and in the Golgi region. Endocytosis by the coated pit/coated vesicle pathway was selectively inhibited by acidification of the cytosol. Under these conditions, both the uptake of toxin-HRP conjugates and intoxication of the cells were inhibited. Evidence from the literature as well as our own results suggest that Shiga toxin binding sites are glycolipids. Thus, Shiga toxin appears to be the first example of a lipid-binding ligand that is endocytosed from coated pits.     

Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA when microinjected into Xenopus oocytes

Ricin, Shiga toxin, and Shiga-like toxin II (SLT-II, Vero toxin 2) exhibit an RNA N-glycosidase activity which specifically removes a single base near the 3' end of 28 S rRNA in isolated rat liver ribosomes and deproteinized 28 S rRNA (Endo Y., Mitsui, K., Motizuki, M., & Tsurugi, K. (1987) J. Biol. Chem. 262, 5908-5912; Endo Y. & Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130, Endo, Y., Tsurugi, K., Yutsudo, T., Takeda, Y., Ogasawara, K. & Igarashi, K. (1988) Eur. J. Biochem. 171, 45-50). These workers identified the single base removed, A-4324, by examining a 28 S rRNA degradation product which was generated by contaminating ribonucleases associated with the ribosomes. To determine whether this N-glycosidase activity applies in living cells, we microinjected ricin into Xenopus oocytes. We also microinjected Shiga toxin and a variant of Shiga-like toxin II (SLT-IIv). All three toxins specifically removed A-3732, located 378 nucleotides from the 3' end of 28 S rRNA. This base is analogous to the site observed in rat 28 S rRNA for ricin, Shiga toxin, and SLT-II. Purified, glycosylated, ricin A chain contains this RNA N-glycosidase activity in oocytes. We also demonstrated that the nonglycosylated A subunit of recombinant ricin exhibits this RNA N-glycosidase activity when injected into Xenopus oocytes. Ricin, Shiga toxin, and SLT-IIv also caused a rapid decline in oocyte protein synthesis for nonsecretory proteins.     

Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits.

The A-subunit polypeptides of Shiga toxin, the Shiga-like toxins (SLTs), and the plant lectin ricin inactivate eucaryotic ribosomes by enzymatically depurinating 28S rRNA. Comparison of the amino acid sequences of the members of the Shiga toxin family and ricin revealed two regions of significant homology that lie within a proposed active-site cleft of the ricin A chain. In previous studies, these conserved sequences of the SLT-I and ricin A subunits have been implicated as active sites. To establish the importance of these regions of homology, we used site-directed mutagenesis to alter the A-subunit sequences of two members of the Shiga toxin family. Substitution of an aspartic acid for glutamic acid 166 of the Slt-IIA subunit decreased the capacity of the polypeptides to inhibit protein synthesis at least 100-fold in a cell-free translation system. However, this mutation did not prevent the expression of immunoreactive, full-length Slt-IIA. In addition, SLT-II holotoxin containing the mutated A subunit was 1,000-fold less toxic to Vero cells. Finally, site-directed mutagenesis was used to delete sequences encoding amino acids 202 through 213 of the Shiga toxin A subunit. Although this deletion did not prevent holotoxin assembly, it abolished cytotoxic activity.     

Shiga toxin B-subunit binds to the chaperone BiP and the nucleolar protein B23

BACKGROUND INFORMATION: In many cell lines, such as HeLa cells, STxB (Shiga toxin B-subunit) is transported from the plasma membrane to the ER (endoplasmic reticulum), via early/recycling endosomes and the Golgi apparatus, bypassing the late endocytic pathway. In human monocyte-derived macrophages and dendritic cells that are not sensitive to Shiga toxin-induced protein biosynthesis inhibition, STxB is not detectably targeted to the retrograde route and is degraded in late endosomes/lysosomes. RESULTS: We have identified B-subunit interacting proteins in HeLa cells and macrophages. In HeLa cells, the ER-localized chaperone BiP (binding protein) was co-immunoprecipitated with the B-subunit. This interaction was not observed in macrophages, consistent with our previous trafficking results. In both cell types, the B-subunit also interacted with the nucleolar protein B23. Consistently, the B-subunit could be detected on nucleoli, suggesting that it could serve to bring the holotoxin to the site of synthesis of its molecular target, rRNA. The nucleolar localization data are critically discussed. CONCLUSION: The interaction of STxB with BiP, involved in the retrotranslocation process to the cytosol and nucleolar B23, as described in this study, might be of relevance for explaining the efficiency of even low doses of Shiga toxin to inactivate cellular ribosomes, and for the use of STxB as a vector for targeting antigens to cytosolic proteasomes of the MHC I-restricted antigen presentation pathway.     

Endocytosis and intracellular transport of the glycolipid-binding ligand Shiga toxin in polarized MDCK cells

The glycolipid-binding cytotoxin produced by Shigella dysenteriae 1, Shiga toxin, binds to MDCK cells (strain 1) only after treatment with short-chain fatty acids like butyric acid or with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. The induced binding sites were found to be functional with respect to endocytosis and translocation of toxin to the cytosol. Glycolipids that bind Shiga toxin appeared at both the apical and the basolateral surface of polarized MDCK cells grown on filters, and Shiga toxin was found to be endocytosed from both sides of the cells. This was demonstrated by EM of cells incubated with Shiga-HRP and by subcellular fractionation of cells incubated with 125I-labeled Shiga toxin. The data indicated that toxin molecules are endocytosed from coated pits, and that some internalized Shiga toxin is transported to the Golgi apparatus. Fractionation of polarized cells incubated with 125I-Shiga toxin showed that the transport of toxin to the Golgi apparatus was equally efficient from both poles of the cells. After 1-h incubation at 37 degrees C approximately 10% of the internalized toxin was found in the Golgi fractions. The results thus suggest that glycolipids can be efficiently transported to the Golgi apparatus from both sides of polarized MDCK cell monolayers.     

Passive protection of purified yolk immunoglobulin administered against Shiga toxin 1 in mouse models

Shiga toxins produced by Escherichia coli O157:H7 cause a wide spectrum of enteric diseases, such as lethal hemorrhagic colitis and hemolytic uremic syndrome. In this study, the B subunit protein of Shiga toxin type 1 (Stx1) was produced in the E. coli system, was further purified by Ni-column Affinity Chromatography method, and was then used as an immunogen to immunize laying hens for yolk immunoglobulin (IgY) production. Titers of IgY increased gradually with boosting vaccination and, finally, reached a level of 105, remaining steady over 1 year. Then the protective efficacy of IgY against Stx1 was evaluated by in vitro and in vivo experiments. It was shown that the anti-Stx1 IgY could effectively block the binding of Stx1 to the Hela cells and could protect BALB/c mice from toxin challenges. The data indicates the facility of using egg yolk IgY as a therapeutic intervention in cases of Shiga toxin intoxication.     

Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan

Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals. To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants. We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount. The 120 strains of Shiga toxin-producing E. coli used in this study were isolated from humans in Japan between 1986 and 1999. Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype. G1 is considered the most important Stx2 variant group in terms of human pathogenicity. A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx.     

Investigation of ribosome binding by the Shiga toxin A1 subunit, using competition and site-directed mutagenesis.

The enzymatic subunit of Shiga toxin (StxA1) is a member of the ribosome-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depurinates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the acceptor site of eukaryotic ribosomes. The specific activities of native StxA1, as well as mutated forms of the enzyme with substitutions in catalytic site residues, were measured by an in vitro translation assay. Electroporation was developed as an alternative method for the delivery of purified A1 polypeptides into Vero cells. Site-directed mutagenesis coupled with N-bromosuccinimide modification indicated that the sole tryptophan residue of StxA1 is required for binding it to the 28S rRNA backbone. Northern analysis established that the catalytic site substitutions reduced enzymatic activity by specifically interfering with the capacity of StxA1 to depurinate 28S rRNA. Ribosomes were protected from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior to binding and depurinating the GAGA tetra-loop.