Anton Glück

Ribotoxin recognition of ribosomal RNA and a proposal for the mechanism of translocation.

The ribotoxins alpha-sarcin and ricin catalyse covalent modifications in adjacent nucleotides in 28S rRNA, yet the elements of nucleic acid structure that they recognize are not only different but incompatible. This suggests that this ribosomal domain (which in two dimensions is a seven-base-pair helical stem and a 17-member single-stranded loop) has alternate conformers. Since the domain is involved in binding of aminoacyl-tRNA and GTP hydrolysis, we propose that the switch between the two configurations, perhaps initiated by the binding of elongation factors, plays a role in translocation.

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 primary structure of rat ribosomal proteins P0, P1, and P2 and a proposal for a uniform nomenclature for mammalian and yeast ribosomal proteins

The covalent structures of rat ribosomal proteins P0, P1, and P2 were deduced from the sequences of nucleotides in recombinant cDNAs. P0 contains 316 amino acids and has a molecular weight of 34,178; P1 has 114 residues and a molecular weight of 11,490: and P2 has 115 amino acids and a molecular weight of 11,684. The rat P-proteins have a near identical (16 of 17 residues) sequence of amino acids at their carboxyl termini and are related to analogous proteins in other eukaryotic species. A proposal is made for a uniform nomenclature for rat and yeast ribosomal proteins.

Dependence of depurination of oligoribonucleotides by ricin A-chain on divalent cations and chelating agents

Ricin A‐chain is a cytotoxic RNA N‐glycosidase that inactivates eukaryotic ribosomes by depurinating the adenosine at position 4324 in 28S rRNA. The enzyme retains its specificity when a synthetic oligoribonucleotide (a 35‐mer) that mimics the structure at the site of action is the substrate. However, covalent modification by ricin A‐chain of the oligoribonucleotide but not of ribosomes, depends on the simultaneous presence of a divalent cation and a chelating agent.

24 Mammalian Ribosomes: The Structure and the Evolution of the Proteins

Ribosomes are universal, essential, and complicated; they mediate protein synthesis in all organisms in our biosphere and thereby link genotype to phenotype. The imperative in research on ribosomes is to determine their structure and thus be able to account for their function. This led to the isolation, the characterization, and the determination of the structures of mammalian (rat) ribosomal proteins and nucleic acids (Wool 1979Wool 1986; Wool et al. 1990). Mammalian ribosomes are composed of two subunits that are designated by their sedimentation coefficients: The smaller is 40S and the larger is 60S. The subunits associate—they are held together by noncovalent bonds, perhaps by magnesium salt bridges—and form the functional 80S ribosome. The 40S subunit has a single molecule of RNA, designated 18S rRNA, and 33 proteins, whereas the 60S subunit has three molecules of RNA, designated 5S, 5.8S, and 28S rRNA, and 47 proteins. The covalent structures of the four species of rat rRNAs (5S, 5.8S, 18S, and 28S) have been established (Nazar et al. 1975; Aoyama et al. 1982; Chan et al. 1983, 1984; Torczyn-ski et al. 1983; Hadjiolov et al. 1984), and there are rational proposals for their secondary structures (Chan et al. 1984; Hadjiolov et al. 1984; Wool 1986; Raue et al. 1988). Eighty-two proteins have been isolated from rat ribosomes, and the complete amino acid sequences in 75 of these proteins have been either determined directly from the protein or deduced from the sequences of nucleotides in cDNAs (for references, see Table A1...