A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ

A new enzyme, which we named ribosomal RNA apurinic site specific lyase (RALyase), is described. The protein was found in wheat embryos and has a molecular weight of 50 625 Da. The enzyme specifically cleaves the phosphodiester bond at the 3' side of the apurinic site introduced by ribosome-inactivating proteins into the sarcin/ricin domain of 28S rRNA. The 3' and 5' ends of wheat 28S rRNA at the cleavage site are 5'-GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA-3', demonstrating that the enzyme catalyzes a beta-elimination reaction. The substrate specificity of the enzyme is extremely high: it acts only at the apurinic site in the sarcin/ricin domain of intact ribosomes, not on deproteinized rRNA or DNA containing apurinic sites. The amino acid sequences of five endopeptidase LysC-liberated peptides from the purified enzyme were determined and used to obtain a cDNA sequence. The open reading frame encodes a protein of 456 amino acids, and a homology search revealed a related rice protein. Similar enzyme activities were also found in other plants that express ribosome-inactivating proteins. We believe that RALyase is part of a complex self-defense mechanism.     

The mechanism of action of trichosanthin on eukaryotic ribosomes--RNA N-glycosidase activity of the cytotoxin.

Trichosanthin is a ribosome-inactivating protein from root tubers of Trichosanthes kirilowii Maxim. In this paper, the mechanism of action of trichosanthin on eukaryotic ribosomes was studied. A fragment of about 450 nucleotides was released from 28S ribosomal RNA after treatment of rat liver ribosome with trichosanthin and its isolated ribosomal RNAs were treated with aniline. Analysis of nucleotide sequence of 5' terminus of this fragment revealed that the aniline-sensitive site of the phosphodiester bond was between positions A4324 and G4325 in the 28S rRNA. Adenine was recovered by ion-exchange column chromatography from the 50% ethanol soluble fraction of the reaction mixture in which rat liver ribosomes were treated with trichosanthin. Thin-layer chromatographic analysis indicated that 1 mol of adenine was released from 1 mol of ribosomes. When the ribosomes were incubated with trichosanthin in the presence of inorganic [32P]phosphate, little incorporation of radioactivity into 28S rRNA was observed, indicating that the release of adenine was not mediated by phosphorolysis. These results demonstrate that trichosanthin inactivates the ribosomes by cleaving the N-C glycosidic bond of adenylic acid at 4324 of 28S rRNA in a hydrolytic fashion.     

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes: the RNA N-glycosidase activity of the proteins

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes was studied. Treatment of ribosomes with any one of these proteins caused the 28S rRNA extracted from the inactivated ribosomes to become sensitive to treatment with aniline. A fragment containing about 450 nucleotides was released from the 28S rRNA. Further analysis of the nucleotide sequences of the 450-nucleotide fragments revealed that the aniline-sensitive phosphodiester bond was between A-4324 and G-4325 of the 28S rRNA. These results indicate that all six ribosome-inactivating proteins damage eukaryotic ribosomes by cleaving the N-glycosidic bond at A-4324 of the 28S rRNA of the ribosomes, as does ricin A-chain.     

Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.     

Ribosome-inactivating proteins from plant cells in culture.

1. Ribosome-inactivating proteins were found in high amounts in one line of cells of Phytolacca americana (pokeweed) cultured in vitro and, in less quantity, in lines of Saponaria officinalis (soapwort) and of Zea mays (corn) cells. 2. The main ribosome-inactivating protein from pokeweed cells was purified to homogeneity. It is a protein with Mr 29,000 and basic pI, similar to the 'pokeweed antiviral protein' (PAP), a ribosome-inactivating protein from pokeweed leaves. We propose to call the pokeweed antiviral protein isolated from pokeweed cells PAP-C. 3. PAP-C inactivates ribosomes in a less-than-equimolar ratio, thus inhibiting protein synthesis by a rabbit reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 0.067 nM (2 ng/ml), and modifies rRNA in a manner apparently identical to that of ricin and other ribosome-inactivating proteins. It inhibits protein synthesis by intact cells with an IC50 of 0.7-3.4 microM, and is toxic to mice with an LD50 of 0.95 mg/kg.     

Resistance of both the 5'- and 3'-domain of isolated Escherichia coli 23S rRNA against digestion with alpha-sarcin

The ribonuclease alpha-sarcin exclusively cleaves the phosphodiester bond after G2661 in the 23S rRNA within 50S subunits, thus inactivating the ribosomes. The resulting alpha-fragment is 243 nucleotides long and contains the 3'-end of the 23S rRNA. The specificity is changed dramatically if isolated 23S rRNA is used as substrate. We have shown previously that 23S rRNA is digested completely except for two fragments, one of which is identical to the alpha-fragment. Here we show that the other fragment comprises the 5'-end of 23S rRNA and contains 385 nucleotides. A similar fragment was obtained when isolated 23S rRNA was digested with RNase A (specific for pyrimidines in single strands). It appears that the 5'-domain (equivalent to 5.8S rRNA of eukaryotic ribosomes) as well as the 3'-domain (equivalent to 4.5S rRNA of chloroplast ribosomes) have a compact and defined tertiary structure in isolated 23S rRNA in contrast to the rRNA region in between. Thus, alpha-sarcin is a convenient tool for detecting compact domains in isolated RNA.     

Type 1 ribosome-inactivating proteins depurinate plant 25S rRNA without species specificity.

Four different type 1 ribosome-inactivating proteins (RIPs) with RNA N-glycosidase activity were tested for their ability to attack the large rRNA of plant ribosomes derived from tobacco plants, as well as from the plant species from which the particular RIP had been isolated. Incubation of tobacco ribosomes with RIPs isolated from either Phytolacca americana L. (pokeweed), Dianthus barbatus L. (carnation), Spinacia oleracea L. (spinach) or Chenopodium amaranthicolor Coste and Reyn. (chenopodium) rendered the 25S rRNA sensitive to aniline-catalyzed hydrolysis, generating a single rRNA-fragment of about 350 nucleotides. The same fragment was generated when rRNAs from pokeweed, carnation, spinach or chenopodium ribosomes were aniline-treated without any deliberate treatment of the ribosomes with the respective RIP. This indicated that ribosomes from all RIP-producing plants were already inactivated by their own RIPs during preparation. These results demonstrate that plant ribosomes are generally susceptible to RIP attack, including modification by their own RIPs. Direct sequencing of the newly generated fragments revealed that a single N-glycosidic bond at an adenosine residue within the highly conserved sequence 5'-AGUACGAGAGGA-3' was cleaved by all of the RIPs investigated, a situation also found in animal, yeast and Escherichia coli ribosomes.     

Functional Escherichia coli 23S rRNAs containing processed and unprocessed intervening sequences from Salmonella typhimurium.

We have introduced the intervening sequence (IVS) from 23S rRNA of the rrnD operon of Salmonella typhimurium into the equivalent position of Escherichia coli 23S rRNA. Salmonella typhimurium 23S rRNA is fragmented due to the RNase III-dependent removal of the approximately 100 nt stem-loop structure that comprises the IVS. In this study, we have found that insertion of the S. typhimurium IVS into E. coli 23S rRNA causes fragmentation of the RNA but does not affect ribosome function. Cells expressing the fragmented 23S rRNA exhibited wild-type growth rates. Fragmented RNA was found in the actively translating polysome pool and did not alter the sedimentation profile of ribosomal subunits, 70S ribosomes or polysomes. Finally, hybrid 23S rRNA carrying the A2058G mutation conferred high level erythromycin resistance indistinguishable from that of intact 23S rRNA carrying this mutation. These observations indicate that the presence of this IVS and its removal are phenotypically silent. As observed in an RNase III-deficient strain, processing of the IVS was not required for the production of functional ribosomes.     

A photolabile oligodeoxyribonucleotide probe of the peptidyltransferase center: identification of neighboring ribosomal components

In this work we report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring its target site in 23S rRNA. The probe is complementary to 23S rRNA nucleotides 2497-2505, a single-stranded sequence that has been shown to fall within the peptidyltransferase center of Escherichia coli ribosomes [Cooperman, B. S., Weitzmann, C. J., & Fernandez, C. L. (1990) in The Ribosome: Structure, Function, & Evolution (Hill, W. E., Dahlberg, A., Garrett, R. A., Moore, P. B., Schlesinger, D., & Warner, J. R., Eds.) pp 491-501, American Society of Microbiology, Washington]. On photolysis in the presence of 50S ribosomes, it site-specifically incorporates into protein L3 (identified by both SDS-PAGE and immunological methods) and into three separate 23S rRNA regions: specifically, nucleotides 2454; 2501, 2502, 2505, 2506; and 2583, 2584. These results provide clear evidence that G-2505 in 23S rRNA is within 24 A (the distance between G-2505 and the photogenerated nitrene) of protein L3 and of each of the nucleotides mentioned above and are of obvious importance in the construction of detailed three-dimensional models of ribosomal structure. The approach we present is general and can be applied to determining ribosomal components neighboring regions of rRNA that are susceptible to binding by complementary oligodeoxyribonucleotides, both in intact 30S and 50S subunits and in subunits at various stages of reconstitution.     

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A⁴³²⁴ in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes.Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.     

The mechanism of action of barley toxin: a type 1 ribosome-inactivating protein with RNA N-glycosidase activity

In a previous report (Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130) it was shown that the RNA N-glycosidase activity of ricin A-chain was responsible for the ability of this protein to inactivate eukaryotic ribosomes. The objective of the present study was to determine whether a similar mechanism was used by a ribosome-inactivating protein from pearled barley (barley toxin). Rat liver ribosomes were incubated either with ricin A-chain or barley toxin, and the rRNA was extracted and treated with acidic aniline to hydrolyze phosphodiester bonds rendered susceptible by removal of a purine or pyrimidine base. Evaluation of the rRNA by polyacrylamide/agarose electrophoresis disclosed two 28 S rRNA-derived fragments which differed in size from those generated by untreated (control) ribosomes. Sequencing of the smaller of these fragments confirmed that - as is the case for ricin A-chain - the aniline-sensitive site in barley toxin-treated ribosomes was between A and G in 28 S rRNA. We conclude that barley toxin inactivates ribosomes via a mechanism identical to that of ricin A-chain: enzymatic hydrolysis of the N-glycosidic bond at A of 28 S rRNA.     

Isolation and partial characterization of nigrin b,a non-toxic novel type 2 ribosome-inactivating protein from the bark ofSambucus nigra L.

The bark ofSambucus nigra L. contains a non-toxic novel type 2 ribosome-inactivating protein that we named nigrin b.In vitro, nigrin b strongly inhibited mammalian protein synthesis but did not affect plant nor bacterial protein synthesis. The protein (M _r 58 000) contains two subunits, A (M _r 26 000) and B (M _r 32 000); linked by disulphide bridge(s). Nigrin b was found to be an rRNA N-glycosidase of the rRNA of intact mammalian ribosomes and shares a very good N-terminal amino-acid sequence homology with the anti-HIV-1 proteins TAP 29 and trichosanthin.     

Wheat ribosome-inactivating proteins: Seed and leaf forms with different specificities and cofactor requirements

Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain VII which has previously been identified as the site of action of ricin A-chain. Tritin-S and tritin-L differ in both their ribosome substrate specificities and cofactor requirements. Tritin-S shows only barely detectable activity on ribosomes from the endosperm, its tissue of synthesis, whereas tritin-L is highly active on leaf ribosomes. Additionally, tritin-S is inactive on wheat germ, tobacco leaf and Escherichia coli ribosomes but active on rabbit reticulocyte and yeast ribosomes. Tritin-L is active on ribosomes from all of the above sources. Tritin-S, unlike tritin-L shows a marked requirement for ATP in its action.Abbreviations CM carboxymethyl - FPLC fast protein liquid chromatography - NEPHGE non-equilibrium pH gradient gel electrophoresis - PAP pokeweed antiviral protein - RIP ribosome-inactivating protein A.J.M. was the recipient of a U.K. Science and Engineering Research Council CASE studentship sponsored by Agricultural Genetics Company Ltd., Cambridge CB4 4GG, UK.     

Colicin E3 is an endonuclease.

It was confirmed by polyacrylamide gel electrophoresis that isolated 16S rRNA was cleaved by the active component (protein A) or the active fragment (T2A) of colicin E3. However, the degradation was random, in contrast with the specific cleavage observed in the interaction of colicin E3 with ribosomes. Furthermore, the active component and the active fragment had low activities, and far greater amounts of these materials were required for degradation of the isolated rRNA than for ribosome inactivation. The degradation of rRNA cannot be due to contaminating ribonuclease(s), but is due to colicin E3 itself, because of the following facts. (1) Protein B of colicin E3, which specifically inhibits the ribosome-inactivating activity of colicin E3, inhibited the degradation of rRNA. (2) Protein B of colicin E2, which inhibits the action of colicin E2 but not of colicin E3, failed to inhibit the degradation of rRNA. (3) The activity appeared in the peak of protein A or fragment T2A, respectively, when they were rechromatographed on Sephadex G-75.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

RIP-JIP60 alters conformation of ribosomes in vivo.

Recently it has been demonstrated that expression putative ribosome-inactivating protein JIP 60 in transgenic tobacco plants leads to the depurination of large rRNA and inactivation of plant ribosomes. Treatment of ribosomes from transgenic tobacco plants with low concentration of alpha-sarcin resulted in the appearance of an rRNA specific fragment. No fragment was observed under the same conditions for ribosomes from wild type plants. The alteration of the comformation of ribosomes in transgenic tobacco due to the expression of JIP60 is assumed.     

Isolation of ribosomal subparticles from human placenta containing intact rRNA and determination of the functional activity of the 80S ribosome

The method for isolation of human placenta ribosomal subunits containing intact rRNA has been determined. The method uses fresh unfrozen placenta. Activity of 80S ribosomes obtained via reassociation of 40S and 60S subunits in non-enzymatic poly(U)-mediated Phe-tRNAPhe binding, was near 75% (maximal [14C]Phe-tRNA(Phe) binding was 1.5 mol Phe-tRNA(Phe) per mol of 80S ribosomes). Activity of 80S ribosomes with damaged rRNA isolated from frozen placenta was 2 times lower (the maximum level of poly(U)-dependent Phe-tRNA(Phe) binding was 0.7 mol per mol of ribosomes). The activity 80S ribosomes in poly(U)-mediated synthesis of polyphenylalanine was determined by using fractionated ("ribosomeless") protein synthesising system from rabbit reticulocytes. In this system up to the 50 mol of Phe residues per mol of 80S ribosomes are incorporated in acid insoluble fraction in 1 hour, at 37 degrees C. The obtained level of [14C]phenylalanine incorporation is three times as much as the amount of Phe residues observed for the ribosomal subunits, isolated from frozen placenta.     

Euglena gracilis chloroplast ribosomal RNA transcription units. Nucleotide sequence polymorphism in 5 S rRNA genes and 5 S rRNAs

The three tandemly repeated ribosomal RNA operons from the chloroplast genome of Euglena gracilis Klebs, Pringsheim Strain Z each contain a 5 S rRNA gene distal to the 23 S rRNA gene (Gray, P.W., and Hallick, R.B. (1979) Biochemistry 18, 1820-1825). We have cloned two distinct 5 S rRNA genes, and determined the DNA sequence of the genes, their 5'- and 3'-flanking sequences, and the 3'-end of the adjacent 23 S rRNA genes. The two genes exhibit sequence polymorphism at five bases within the "procaryotic loop" coding region, as well as internal restriction endonuclease site heterogeneity. These restriction endonuclease site polymorphisms are evident in chloroplast DNA, and not just the cloned examples of 5 S genes. Chloroplast 5 S rRNA was isolated, end labeled, and sequenced by partial enzymatic degradation. The same polymorphisms found in 5 S rDNA are present in 5 S rRNA. Therefore, both types of 5 S rRNA genes are transcribed and are present in chloroplast ribosomes.     

The solution structure of the loop E region of the 5S rRNA from spinach chloroplasts

A structure has been obtained for the loop E region of the 5S rRNA from Spinacia oleracia chloroplast ribosomes using residual dipolar coupling data as well as NOE, J coupling and chemical shift information. Even though the loop E sequence of this chloroplast 5S rRNA differs from that of Escherichia coli loop E at approximately 40% of its positions, its conformation is remarkably similar to that of E.coli loop E. Consistent with this conclusion, ribosomal protein L25 from E.coli, which binds to the loop E region of both intact E.coli 5S rRNA and to oligonucleotides containing that sequence, also binds to the chloroplast-derived oligonucleotide discussed here.