Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.     

Evidence that Escherichia coli 30 S ribosomal subunit populations are conformationally heterogeneous.

We have estimated the number of sites on each protein of the 30 S ribosome which are accessible to chemical iodination. First, the total number of iodinatable sites was determined for the intact 30 S ribosome. The proteins were extracted, separated and the relative distribution of iodine in each protein determined. This distribution of iodine divided into the total sites per ribosome gave an estimate of the number of sites per individual protein.Second, the iodinated proteins were purified and their trypsin digestion products separated. The number of radioactive peptides was taken as a measure of the number of sites on that protein open to the iodination reaction. The number of iodinatable sites for each protein was found to be radically different by the two methods. In almost all cases, the number of unique, radioactively labeled peptides, derived from a given 30 S protein, far exceeded the total incorporation into that protein. We suggest that the best explanation for this unexpected discrepancy is that the 30 S ribosome population we used in these experiments is heterogeneous in its topography.In addition we have compared the topography by the chemical iodination procedure for ribosomes in two different conformations: active and inactive (see Zamir et al., 1971). We have found very little change in the chemical reactivity of the proteins when the ribosomes are in the two different conformations. The most notable changes involve proteins S10, $\text{S}\text{18}\text{S}\text{19}$ and especially $\text{S}\text{12}\text{S}\text{13}$.     

Effects of aminoethylation of cysteine residues on the structural and functional activities of the Escherichia coli 30 S ribosomal proteins

The purified 30 S ribosomal proteins from Escherichia coli strain Q13 were chemically modified by reaction with ethyleneimine, specifically converting cysteine residues to S-2-aminoethylcysteine residues. Proteins S1, S2, S4, S8, S11, S12, S13, S14, S17, S18 and S21 were found to contain aminoethylcysteine residues after modification, whereas proteins S3, S5, S6, S7, S9, S10, S15, S16, S19 and S20 did not. Aminoethylated proteins S4, S13, S17 and S18 were active in the reconstitution of 30 S ribosomes and did not have altered functional activities in poly(U)-dependent polyphenylalanine synthesis, R17-dependent protein synthesis, fMet-tRNA binding and Phe-tRNA binding. Aminoethylated proteins S2, S11, S12, S14 and S21 were not active in the reconstitution of complete 30 S ribosomes, either because the aminoethylated protein did not bind stably to the ribosome (S2, S11, S12 and S21) or because the aminoethylated protein did not stabilize the binding of other ribosomal proteins (S14). The functional activities of 30 S ribosomes reconstituted from a mixture of proteins containing one sensitive aminoethylated protein (S2, S11, S12, S14 or S21) were similar to ribosomes reconstituted from mixtures lacking that protein. These results imply that the sulfhydryl groups of the proteins S4, S13, S17 and S18 are not necessary for the structural or functional activities of these proteins, and that aminoethylation of the sulfhydryl groups of S2, S11, S12, S14 and S21 forms either a kinetic or thermodynamic barrier to the assembly of active 30 S ribosomes in vitro.     

Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 50s ribosomal subunits or 70s ribosomes

Further to our studies on the Escherichia coli 30S ribosomal subunit, the detailed surface topography of both 50S subunits and 70S ribosomes has been investigated by using iodination catalyzed by immobilized lactoperoxidase as the surface probe. In the 50S subunit, only proteins L2, L5, L10, and L11 were iodinated to a significant and reproducible extent. The targets of iodination were identified, after isolation of the individual iodinated proteins, and were as follows: in protein L2 (271 amino acids), tyrosine-102 and -160; in protein L5 (178 amino acids), tyrosine-142; in protein L10 (165 amino acids), tyrosine-132; in protein L11 (142 amino acids), tyrosine-7 and -61. In the 70S ribosome, only protein L5 was still iodinated to a significant extent from the 50S subunit, whereas in the 30S subunit the same spectrum of iodinated proteins was observed as that from iodinated isolated 30S subunits, with the exception that S21 was no longer present.     

Preliminary x-ray diffraction data for tetragonal crystals of trypsinized Escherichia coli elongation factor.

Previous studies (Craven et al., 1974) demonstrated that the capacity of ribosomal proteins to be chemically modified by iodine is extensively reduced when they are members of an intact ribosome. We have attempted to exploit this observation by analyzing in detail the alterations in the iodine accessibility of the individual 30 S ribosomal proteins. We have prepared a total of 38 different complexes between 16 S RNA and mixtures of individual purified 30 S ribosomal proteins. Eighteen of the 21 30 S proteins were used in the formation of these complexes. Comparison of the iodination patterns obtained for the various proteins derived from different complexes has revealed that sometimes a specific protein can selectively alter the chemical reactivity of another protein in the complex. We have found 30 different examples of protein pairs in which one protein effectively protects another protein from chemical iodination.     

The ribosomal binding domain of the Escherichia coli release factors. Modification of tyrosine in the N-terminal domain of ribosomal protein L11 affects release factors 1 and 2 differentially

Ribosomal protein L11 is one of only two ribosomal proteins significantly iodinated when Escherichia coli 50 S subunits are modified by immobilized lactoperoxidase, and the major target has been shown previously to be tyrosine at position 7 in the N-terminal domain. This modification reduces in vitro termination activity with release factor (RF)-1 by 70-90%, but RF-2 activity is less affected (30-50%). The loss of activity parallels incorporation of iodine into the subunit. The 50 S subunits from L11-lacking strains of bacteria have highly elevated activity with RF-2 and low activity with RF-1. The iodination does not affect RF-2 activity but reduces the RF-1 activity further. Ribosomal proteins, L2, L6, and L25, are significantly labeled in L11-lacking ribosomes in contrast to the control 50 S subunits. L11 has been modified in isolation and incorporated back efficiently into L11-lacking ribosomes. This L11, iodinated also predominantly at Tyr 7, is unable to restore RF-1 activity to L11-lacking ribosomes in contrast to mock-iodinated protein. These results suggest the involvement of the N terminus of L11 in the binding domain of the bacterial release factors and indicate that there are subtle differences in how the two factors interact with the ribosome.     

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.     

The subunit interface of the Escherichia coli ribosome: Identification of proteins at the interface between the 30 S and 50 S subunits by crosslinking with 2-iminothiolane

The 70 S ribosomes of Escherichia coli were treated with 2-iminothiolane with the resultant addition of 110 sulfhydryl groups per ribosome. The modified ribosomes were oxidized to promote disulfide bond formation, some of which formed intermolecular crosslinks. About 50% of the crosslinked 70 S ribosomes did not dissociate when exposed to low concentrations of magnesium in the absence of reducting agent. Dissociation took place in the presence of reducing agents, which indicated that the subunits had become covalently linked by disulfide linkages. Proteins extracted from purified crosslinked 70 S ribosomes were first fractionated by polyacrylamide/urea gel electrophoresis. The proteins from sequential slices of these gels were analyzed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Monomeric proteins derived from crosslinked dimers appeared below the diagonal containing non-crosslinked proteins, since the second electrophoresis, but not the first, is run under reducing conditions to cleave the crosslinked species. Final identification of the proteins in each dimer was made by radioiodination of the crosslinked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of non-radioactive total 70 S proteins as markers. This paper describes the identification of 23 protein dimers that contained one protein from each of the two different ribosomal subunits. The proteins implicated must have some part of their structure in proximity to the other ribosomal subunit and are therefore defined as “interface proteins”. The group of interface proteins thus defined includes 50 S proteins that are part of the 5 S RNA: protein complex and 30 S proteins at the initiation site. Correlations between the crosslinked interface proteins and other functional data are discussed.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precursor pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [³⁵S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s.The [³⁵S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30–120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation.Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Identification of neighbor relationships among proteins in the 30 S ribosome: intermolecular cross-linkage of three proteins induced by tetranitromethane

Earlier studies have indicated that the reaction of tetranitromethane with the 30 S riboaome from Escherichia coli results in the disappearance of two protein bands from the polyacrylamide gel electrophoresis pattern (Craven et al., 1969b). As tetranitromethane is known to induce intermolecular cross-linkage in other protein systems, we studied further this reaction with the view that it might yield knowledge of protein-protein neighbor relationships within the ribosome.The use of two-dimensional polyacrylamide gel electrophoresis showed that the reaction with tetranitromethane caused the disappearance of four proteins from the pattern of 30 S ribosomal proteins. It was shown that this alteration in electrophoretic behavior was not due to simple protein modification (e.g. production of 3-nitrotyrosine), as reaction with extracted protein in 8 M-urea resulted in no observable change in the electrophoretic pattern.It was also shown that three of these proteins could be uniquely labeled with [¹⁴C]iodoacetate without changing their reactivity with tetranitromethane. Thus, ribosomes were labeled with [¹⁴C]iodoacetate, reacted with tetranitromethane and the radioactive reaction products were isolated by column chromatography and preparative gel electrophoresis. The radioactive peptide patterns of the three proteins digested by trypsin were compared with the three major reaction products. One of these products was shown to contain the radioactive tryptic peptides of all three proteins. We believe that this reaction product is an intermolecular cross-linked aggregate of these three proteins, identified as S11, S18 and S21. We suggest that these three proteins are clustered closely together in the 30 S ribosome. The fourth protein, S12, may also be involved in this aggregate.     

Further evidence for the participation of proteins S 3, S 14 and S 19 in tRNA binding to E. coli 30 S subunits

Previous studies have shown that iodination of 30 S subunits causes inactivation for both enzymatic fMet-tRNA and non-enzymatic phe-tRNA binding activities. This inactivation was shown to be due to the modification of three to five ribosomal proteins [1]. In this report the role of these proteins in tRNA binding activity has been further studied. Purified ribosomal proteins, isolated from modified subunits, are re-assembled into otherwise unmodified 30 S ribosomes and assayed for tRNA binding capacity. The presence of modified S 3, S 14 and S 19 (S 15) in the reconstituted particle results in substantial reduction of both fMet-tRNA and phe-tRNA binding activities. This reduction in tRNA binding activity does not appear to be due to an assembly defect.     

Mapping proteins of the 50S subunit from Escherichia coli ribosomes

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.     

Characterization of different conformational forms of 30 S ribosomal subunits in isolated and associated states: possible correlations between structure and function.

The reaction pattern with N-[¹⁴C]ethylmaleimide served to follow conformational changes of 30 S ribosomal subunits that are induced by association with 50 S subunits and by the binding of aminoacyl-tRNA to 70 S ribosomes either enzymatically or non-enzymatically.The usefulness of the reaction with N-ethylmaleimide in discerning different conformational forms of the ribosome was previously demonstrated (Ginzburg et al., 1973) in an analysis of inactive and active 30 S subunits (as obtained at low Mg²⁺ and after heat reactivation, respectively). The reaction pattern of the 30 S moiety of 70 S ribosomes differs from the pattern of isolated active subunits (the only form capable of forming 70 S ribosomes) in both the nature of the labeled proteins and in being Mg²⁺-dependent. The reaction at 10 mm-Mg²⁺ reveals the following differences between isolated and reassociated 30 S subunits: (1) proteins S1, S18 and S21 that are not labeled in isolated active subunits, but are labeled in the inactive subunits, are highly reactive in 70 S ribosomes; (2) proteins S2, S4, S12 and S17 that uniquely react with N-ethylmaleimide in active subunits are all rendered inaccessible to modification after association; and (3) proteins S9, S13 and S19, that react in both active and inactive 30 S subunits, are labeled to a lesser extent in the 70 S ribosomes than in isolated subunits. This pattern is altered in two respects when the reaction with the maleimide is carried out at 20 mm-Mg²⁺; protein S18 is not modified while S17 becomes labeled.The differences in reaction pattern are considered as manifesting the existence of different conformational forms of the 30 S subunit in the dissociated and associated states as well as of different forms of 70 S ribosomes. The 30 S moiety of 70 S ribosomes at 10 mm-Mg²⁺ resembles the inactive subunit, while some of the features of the active subunit are preserved in the 70 S ribosome at 20 mmMg²⁺. The structural changes appear to be expressed in the functioning of the ribosome: non-enzymatic binding of aminoacyl-tRNA to active 30 S subunits is suppressed by 50 S subunits at 10 mm but not at 20 mm-Mg²⁺ (Kaufmann &; Zamir, 1972). The fact that elongation factor Tu-mediated binding is not suppressed by 50 S subunits raises the possibility that the function of the elongation factor might involve the facilitation of a conformational change of the ribosome. The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18. Binding of aminoacyl-tRNA, on the other hand, is closely correlated with the exposure of S17 for reaction with the maleimide. A model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu.     

Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins

Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.     

Role of protein L25 and its contact with protein L16 in maintaining the active state of <Emphasis Type="Italic">Escherichia coli</Emphasis> ribosomes <Emphasis Type="Italic">in vivo</Emphasis>

A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Photolabeling of protein components in the pactamycin binding site of rat liver ribosomes

The antitumoral and antibacterial drug pactamycin can be radioactively labeled by iodination without loss of biological activity. Using the labeled pactamycin, the ribosomal binding site of the drug on rat liver ribosomes has been studied by affinity labeling techniques taking advantage of the photoreactive acetophenone group present in the molecule. When 40 S ribosomal subunits are labeled, one major spot of radioactivity is found associated to protein S25. In addition, weaker spots related to proteins S14/15, S10, S17 and S7 can also be detected in the autoradiogram of the two-dimensional gel slab. Since pactamycin inhibits protein synthesis initiation, the proteins forming its binding site must be related to some step of this process. By comparison with results from pactamycin affinity labeling of Escherichia coli ribosomes (Tejedor, F., Amils, R. and Ballesta, J.P.G. (1985) Biochemistry 24, 3667-3672) these proteins could lie in the mRNA and initiation factors binding region of the rat liver ribosome.     

Multiple defects in translation associated with altered ribosomal protein L4

The ribosomal proteins L4 and L22 form part of the peptide exit tunnel in the large ribosomal subunit. In Escherichia coli, alterations in either of these proteins can confer resistance to the macrolide antibiotic, erythromycin. The structures of the 30S as well as the 50S subunits from each antibiotic resistant mutant differ from wild type in distinct ways and L4 mutant ribosomes have decreased peptide bond-forming activity. Our analyses of the decoding properties of both mutants show that ribosomes carrying the altered L4 protein support increased levels of frameshifting, missense decoding and readthrough of stop codons during the elongation phase of protein synthesis and stimulate utilization of non-AUG codons and mutant initiator tRNAs at initiation. L4 mutant ribosomes are also altered in their interactions with a range of 30S-targeted antibiotics. In contrast, the L22 mutant is relatively unaffected in both decoding activities and antibiotic interactions. These results suggest that mutations in the large subunit protein L4 not only alter the structure of the 50S subunit, but upon subunit association, also affect the structure and function of the 30S subunit.     

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sδ. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A⁵¹ residue on 28Sδ. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.