肌苷(6-脱氨基腺苷)是一种特征性的修饰核苷,在真核和真细菌起源的几种tRNA的第一个反密码子位置(位置34)发现,而N1-甲基肌苷仅在真核tRNA(Ala)的第37位(与反密码子相邻的3')和嗜盐和嗜热古细菌的几种tRNA的第57位(在psi环的中间)发现。最近在双链RNA中也发现了肌苷,mRNA和病毒RNA。至于RNA中所有其他修饰的核苷,在这些RNA中肌苷和肌苷衍生物的形成由RNA基因转录后起作用的特定酶催化。使用几个tRNA基因的重组tRNA和T7径流转录本作为底物,我们已经研究了tRNA-肌苷形成酶的机制和特异性。结果表明,tRNA中的肌苷-34和肌苷-37都是通过水解脱氨反应合成的,由不同的tRNA催化:腺苷脱氨酶。脱氨酶对tRNA底物的识别并不严格取决于特定的“身份”核苷酸。然而,腺苷转化为肌苷的效率取决于反密码子环和近端茎的核苷酸组成以及tRNA的3D结构。在真核tRNA(Ala)中,N1-甲基肌苷37是由肌苷37通过特定的SAM依赖性甲基化酶形成的,而在古细菌tRNA中的N1-甲基肌苷-57的情况下,腺苷-57甲基化为N1-甲基腺苷-57发生在脱氨过程之前。片段化的tRNA的Tpsi分支是N1-甲基肌苷-57形成酶的极简底物。人tRNA(Ala)中的肌苷-34和N1-甲基肌苷-37是特定自身抗体的靶标,这些抗体存在于患有PL-12多发性肌炎类型的炎性肌肉疾病的患者的血清中。在这里我们讨论的机制,最近发现的RNA的特异性和一般特性:作用于双链RNA的腺苷脱氨酶/editase,含有内含子的mRNA和病毒RNA与作用于tRNA的脱氨酶的那些相关。
Inosine (6-deaminated adenosine) is a characteristic modified nucleoside that is found at the first
anticodon position (position 34) of several tRNAs of eukaryotic and eubacterial origins, while N1-methylinosine is found exclusively at position 37 (3\' adjacent to the
anticodon) of eukaryotic tRNA(Ala) and at position 57 (in the middle of the psi loop) of several tRNAs from halophilic and thermophilic archaebacteria. Inosine has also been recently found in double-stranded RNA, mRNA and viral RNAs. As for all other modified nucleosides in RNAs, formation of inosine and inosine derivative in these RNA is catalysed by specific enzymes acting after transcription of the RNA genes. Using recombinant tRNAs and T7-runoff transcripts of several tRNA genes as substrates, we have studied the mechanism and specificity of tRNA-inosine-forming enzymes. The results show that inosine-34 and inosine-37 in tRNAs are both synthesised by a hydrolytic deamination-type reaction, catalysed by distinct tRNA:adenosine deaminases. Recognition of tRNA substrates by the deaminases does not strictly depend on a particular \"identity\' nucleotide. However, the efficiency of adenosine to inosine conversion depends on the nucleotides composition of the
anticodon loop and the proximal stem as well as on 3D-architecture of the tRNA. In eukaryotic tRNA(Ala), N1-methylinosine-37 is formed from inosine-37 by a specific SAM-dependent methylase, while in the case of N1-methylinosine-57 in archaeal tRNAs, methylation of adenosine-57 into N1-methyladenosine-57 occurs before the deamination process. The T psi-branch of fragmented tRNA is the minimalist substrate for the N1-methylinosine-57 forming enzymes. Inosine-34 and N1-methylinosine-37 in human tRNA(Ala) are targets for specific autoantibodies which are present in the serum of patients with inflammatory muscle disease of the PL-12 polymyositis type. Here we discuss the mechanism, specificity and general properties of the recently discovered RNA:adenosine deaminases/editases acting on double-stranded RNA, intron-containing mRNA and viral RNA in relation to those of the deaminases acting on tRNAs.