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

Endo, Yaeta; Glück, Anton; Wool, Ira G.

doi:10.1016/0022-2836(91)80214-f

Cited by 134 publications

(86 citation statements)

References 26 publications

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“…This site of action is a highly conserved loop-and-stem structure, and the conditions necessary for its identification by ricin have been determined by Endo and co-workers [7]. This is the only known site of action of RIPs on eukaryotic ribosomes, although ricin [7] and Mirabilis antiviral protein [3] also depurinated naked 16 S Escherichia coli rRNA at another site, A-1014. We report here that saporins (RIPs from seeds, leaves and roots of Saponaria officinalis), pokeweed antiviral protein from Phytolacca americana roots (PAP-R), and trichokirin from Trichosanthes kirilowii seeds depurinate rat liver ribosomes more extensively than do other known RIPs.…”

Section: Introduci Onmentioning

confidence: 99%

“…They are RNA N-glycosidases which depurinate the major ribosomal RNA at a position corresponding to adenine-4324 of rat liver 28 S rRNA [5,6]. This site of action is a highly conserved loop-and-stem structure, and the conditions necessary for its identification by ricin have been determined by Endo and co-workers [7]. This is the only known site of action of RIPs on eukaryotic ribosomes, although ricin [7] and Mirabilis antiviral protein [3] also depurinated naked 16 S Escherichia coli rRNA at another site, A-1014.…”

Section: Introduci Onmentioning

confidence: 99%

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Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites

Barbieri

Ferreras

Barraco

et al. 1992

Biochemical Journal

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Saporin-S6, a ribosome-inactivating protein (RIP) from Saponaria officinalis released more than 1 mol of adenine/mol of ribosomes from house fly (Musca domestica) larvae and from rat liver. The release of adenine from rat liver ribosomes by several RIPs (plant enzymes with RNA N-glycosidase activity) was examined. Saporins, pokeweed antiviral protein from roots of Phytolacca americana (PAP-R), and trichokirin from Trichosanthes kirilowii seeds depurinated rat liver ribosomes at more than one site. Up to 33 mol of adenine were released from 1 mol of ribosomes. This property is not common to all RIPS. INTRODUCI ONRibosome-inactivating proteins (RIPs) from plants (reviewed in [11), of either type I (single-chain proteins) or type 2 (twochain proteins, ricin and related toxins), damage ribosomes from eukaryotes and, at higher concentrations, ribosomes from prokaryotes 12-4J. They are RNA N-glycosidases which depurinate the major ribosomal RNA at a position corresponding to adenine-4324 of rat liver 28 S rRNA [5,6]. This site of action is a highly conserved loop-and-stem structure, and the conditions necessary for its identification by ricin have been determined by Endo and co-workers [7]. This is the only known site of action of RIPs on eukaryotic ribosomes, although ricin [7] and Mirabilis antiviral protein [3] also depurinated naked 16 S Escherichia coli rRNA at another site, A-1014. We report here that saporins (RIPs from seeds, leaves and roots of Saponaria officinalis), pokeweed antiviral protein from Phytolacca americana roots (PAP-R), and trichokirin from Trichosanthes kirilowii seeds depurinate rat liver ribosomes more extensively than do other known RIPs.

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Section: Introduci Onmentioning

confidence: 99%

Section: Introduci Onmentioning

confidence: 99%

Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites

Barbieri

Ferreras

Barraco

et al. 1992

Biochemical Journal

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“…The identity elements in the ricin domain stem and loop in 28S rRNA are being determined by analyzing the effect of the toxin on mutants of a synthetic RNA that reproduces this structure (6,7). Complementary information for the protein is needed.…”

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confidence: 99%

Determination by systematic deletion of the amino acids essential for catalysis by ricin A chain.

Morris

Wool

1992

Proc. Natl. Acad. Sci. U.S.A.

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“…The target site adenine for RIPs is on a stem-loop structure which protrudes into the acceptor site groove of the large ribosomal subunit (3,4). This is the rRNA domain involved in EF-1-dependent binding of aminoacyl tRNAs to the ribosome and in EF-2-catalyzed GTP hydrolysis and translocation of peptidyl tRNA to the P site.…”

Section: Discussionmentioning

confidence: 99%

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

Skinner

Jackson

1997

J Bacteriol

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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.Shiga toxin (STX) is a multimeric molecule consisting of a single 32-kDa enzymatic A subunit (StxA) noncovalently associated with a ring of five 7.7-kDa B subunits (StxB) (17). The StxB subunits mediate the binding of toxin to its receptor on eukaryotic membranes, and the entire STX molecule enters cells via endocytosis from coated pits (28). StxA is processed in the trans-Golgi network by the cellular protease furin (11) to release StxA1, which possesses the enzymatic activity, and StxA2, which acts as a bridge between StxA1 and the B pentamer. StxA1 is an N-glycosidase which catalytically removes a specific adenine from a well-conserved tetra-loop of 28S rRNA and thus inhibits protein synthesis by preventing the binding of aminoacyl tRNAs to the ribosome and halting protein elongation (7). StxA1 is a member of the ribosome-inactivating protein (RIP) family of enzymes, which are a large group of protein synthesis inhibitors primarily found in the leaves, seeds, and roots of dicotyledonous plants (5,7,30). The only RIPs identified so far which are not produced by plants are the members of the STX family of toxins.The catalytic sites of StxA1 have been identified by sequence comparison of RIP family members and site-specific mutagenesis. Amino acid substitutions in three regions of StxA1 which are highly conserved among the RIPs identified tyrosine 77, glutamic acid 167, arginine 170, and tryptophan 203 as important for catalytic activity (2,16,33). Corresponding mutations in another RIP, the A chain of the plant lectin ricin (RTA), corroborated the findings with StxA1 (24, 29).The X-ray crystal structures of StxA1 and RTA have been solved at 2.5 Å (10, 20). Comparison of these structures revealed an active site cleft which contains all of the catalytic site residues previously identified by site-specific mutagenesis. Xray crystallographic analysis of StxA1 and RTA als...

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

Cited by 134 publications

References 26 publications

Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites

Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites

Determination by systematic deletion of the amino acids essential for catalysis by ricin A chain.

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

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