RNA-protein interaction. An analysis with RNA oligonucleotides of the recognition by alpha-sarcin of a ribosomal domain critical for function

• Published in: The Journal of biological chemistry 1990-02, Vol.265 (4), p.2216-2222
• Main Authors: ENDO, Y, GLUCK, A, YUEN-LING CHAN, TSURUGI, K, WOOL, I. G
• Format: Article
• Language: English
• Subjects: Adenosine; Analytical, structural and metabolic biochemistry; Aspergillus - metabolism; Base Composition; Base Sequence; Biological and medical sciences; Endoribonucleases; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Fungal Proteins - metabolism; Hydrolases; Molecular Sequence Data; Nucleic Acid Conformation; Oligoribonucleotides - metabolism; Protein Binding; Protein Synthesis Inhibitors - metabolism; RNA, Ribosomal, 28S - metabolism; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)39964-8

alpha-Sarcin is a cytotoxic protein that inactivates ribosomes by hydrolyzing a single phosphodiester bond on the 3' side of G-4325 in eukaryotic 28 S rRNA. We have examined the requirements for the recognition by alpha-sarcin of this domain using a synthetic oligoribonucleotide (35-mer) that reproduces the sequence and, we presume, the secondary structure (a stem, a bulged nucleotide, and a loop) at the site of modification. The wild type structure and a large number of variants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. Recognition of the substrate is strongly favored by a G at the position that corresponds to 4325. There is an absolute requirement for a helical stem; however, it can be reduced from the 7 base pairs in the natural structure to 3 without loss of specificity. The nature of the base pairs in the stem modifies but does not abolish recognition; whereas, the bulged nucleotide does not contribute to identification. Cleavage is materially affected by altering the nucleotides in the universal sequence surrounding G-4325 and changing the position in the loop of the tetranucleotide GAG(sarcin)A leads to loss of recognition by the toxin. We propose that the alpha-sarcin domain RNA participates in elongation factor catalyzed binding of aminoacyl-tRNA and of translocation; that translocation is driven by transitions in the structure of the alpha-sarcin domain RNA initiated by the binding of the factors, or the hydrolysis of GTP, or both; and that to toxin inactivates the ribosomes by preventing this transition.