The amino acid sequence of rat ribosomal protein L36a, which may form part of the peptidyl transferase center, was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the amino-terminal amino acid sequence of the protein. Ribosomal protein L36a contains 105 amino acids (the amino-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 12,311. Hybridization of the cDNA to digests of nuclear DNA suggests that there are multiple copies of the L36a gene. Rat ribosomal protein 136a is homologous to a protein HL44 present in ribosomes of humans and protein 44 from Saccharomyces cerevisiae ribosomes.
An oligoribonucleotide (35-mer) that mimics the alpha-sarcin and the ricin region of eukaryotic 28 S rRNA was transcribed in vitro from a synthetic template with T7 RNA polymerase and was used to test whether the specificity of the hydrolysis by the toxins was retained. alpha-Sarcin, at a low concentration, cleaved a single phosphodiester bond on the 3' side of a guanosine residue in the synthetic oligomer that corresponds to G-4325 in 28 S rRNA, the site of action of the toxin in intact ribosomes. At a high concentration of alpha-sarcin, the substrate (35-mer) was hydrolyzed after each of its purines. alpha-Sarcin was without an effect on a synthetic RNA (20-mer) that reproduces the near universal sequence of nucleotides in the loop, but lacks the stem, of the toxin's domain. Thus, the specificity of the attack of alpha-sarcin on a precise region of 28 S rRNA appears to be contingent on the sequence of the nucleotides and the structure of the domain. Ricin depurinated a nucleotide in the synthetic oligomer (35-mer), and in the presence of aniline the phosphoribose backbone was cleaved at a position that conforms to A-4324 in 28 S rRNA, the site of action of the toxin in vivo.
Summary An extracellular protein was isolated from a species of soil‐borne fungi ( Trichoderma viride ) and its amino acid composition has been determined. The protein is acidic with a molecular mass of 14 200 daltons and is given the trivial name tricholin. Tricholin is a potent inhibitor of cell‐free protein synthesis. When rabbit reticulocyte lysate was incubated with tricholin at a concentration of 6.3 × 10 −7 M, it completely abolished the capacity of the lysate to support protein synthesis. The inhibition appears to be due to its reaction to ribosomes, since it generates a specific cleavage product, an α‐sarcin RNA fragment, from reticulocyte ribosomal RNA. This reaction to ribosomes mimics that of α‐sarcin. The antibody of α‐sarcin strongly cross‐reacts with tricholin, while the antibody of tricholin shows a weak reaction with α‐sarcin.
A mutant of ribonuclease T1 (RNase T1), denoted RNase Tα, that is designed to recognize double-stranded ribonucleic acid was created. RNase Tα carries the structure of RNase T1 except for a part of its loop L3 domain, which has been swapped for a corresponding domain from α-sarcin. The RNase Tα maintains the pleated β-sheet structure and retains the guanyl-specific ribonuclease activity of the wild-type RNase T1. A steady-state kinetic study on the RNase Tα-catalyzed transesterification of GpU dinucleoside phosphates reveals a slightly reduced Km value of 6.94×10−7 M. When the stranded specificity is examined, RNase Tα catalyzes the hydrolysis of guanine base not only of single-stranded but also, as by design, of double-stranded RNA. The change of stranded specificity suggests the feasibility of using domain swapping to make a substrate-specific ribonuclease. This study suggests that the loop L3 in RNase T1 can be used as a ‘cassette player’ for inserting a functional domain to make ribonuclease of various specificities.
The substrate specificity of monomeric and dimeric forms of α‐sarcin was investigated by membrane blotting procedures. Dimeric α‐sarcin fails to inactivate ribosomes as well as to hydrolyze mini‐stem‐loop RNA, whereas monomeric α‐sarcin catalyzes both substrates. Both monomeric and dimeric α‐sarcin are effective ribonucleases that are displayed by in situ RNA‐impregnated gel electrophoresis. The same purine base specificity was detected for both dimeric and monomeric forms. α‐Sarcin is also an effective deoxyribonuclease to supercoiled DNA. The action of α‐sarcin as deoxyribonuclease and ribonuclease is inhibited by the presence of SDS (3.5 × 10 −6 M); the inhibition on ribonuclease, but not on deoxyribonuclease, is reversible if the proteins are renatured.
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