Brigotti M, Carnicelli D, Alvergna P, Pallanca A, Lorenzetti R, Denaro M, Sperti S, Montanaro L
Dipartimento di Patologia sperimentale dell'Università di Bologna, Italy.
Biochem J. 1995 Aug 15;310 ( Pt 1)(Pt 1):249-53. doi: 10.1042/bj3100249.
Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3'-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.
相思豆毒素(一种对28S核糖体RNA具有RNA N - 糖苷酶活性的核糖体失活蛋白)对核糖体的失活作用需要核糖体后上清液中存在的大分子辅因子。其中一种辅因子已从大鼠肝脏提取物中纯化出来,鉴定为一种约70个核苷酸长的RNA,序列分析表明它与哺乳动物(牛)的色氨酸转运RNA(tRNA(Trp))具有高度相似性。测序凝胶的图谱与制备物中两种3'-未成熟的tRNA(Trp)物种的共存一致,这两种物种仅缺少A75,或同时缺少A75和C74。在ATP、CTP和tRNA核苷酸转移酶存在的情况下,刺激相思豆毒素活性的RNA是色氨酸的良好受体。如凝胶迁移率变动分析和核糖核酸酶H消化所示,与哺乳动物(牛)tRNA(Trp)的55至72位互补的寡聚脱氧核苷酸与刺激相思豆毒素活性的RNA杂交。寡聚脱氧核苷酸导向的核糖核酸酶H处理也消除了RNA粗制品的相思豆毒素促进活性,有力地证明唯一具有活性的RNA是一种类似tRNA(Trp)的分子。