A ribozyme exclusively aminoacylates the 3'-hydroxyl group of the tRNA terminal adenosine.
A ribozyme exclusively aminoacylates the 3'-hydroxyl group of the tRNA terminal adenosine.
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核酶专门氨酰化 tRNA 末端腺苷的 3-羟基。
DOI：
10.1021/ja015756s
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发表时间：
2001
影响因子：
15
通讯作者：
Suga,H
中科院分区：
文献类型：
--
作者：
Saito,H;Suga,H
基于RNA的氨酰化系统可能在建立古代遗传密码方面发挥了关键作用。1-4在这个系统中，一组催化RNA分子（核酶）应该能够以类似于现代蛋白质氨酰-tRNA合成酶（ARS）的方式氨酰化tRNA（或其原始类似物）。1，4-6我们已经使用体外进化技术从含有1015个独特序列的RNA组合池中分离出这样的ARS样核酶（图1A）。[7]我们报道了这种核酶能够专一地氨酰化tRNA的3′-羟基（3′-OH）。在现代翻译系统中，遗传编码事件是由人工翻译系统完成的。8它们通常以20种不同的形式存在，每一种都催化特定氨基酸与同源tRNA异源受体的3′-末端腺苷（A76）的酯化反应，从而将每个氨基酸连接到其相应的tRNA反密码子三联体。ARS的所有成员在结构上分为两类（I和II），9，10，并且这些类别之间酯化位点的选择是不同的：I类酶氨酰化tRNA上A76的2′-OH，而II类酶氨酰化3′-OH。[11-14]在RNA世界假说的背景下，1，15，16我们已经进化出一种前体tRNA（pre-tRNA），它特异性地将苯丙氨酸（Phe）装载到自己的3′端。[7]这种催化性前tRNA与天然存在的核酸内切酶核酶（RNase P RNA）相容，导致其断裂为5′-前导序列和成熟tRNA（otRNA，17，18图1B）。所得的5′-前导片段能够将Phe装载到otRNA上，从而表现为反式作用的ARS样核酶（称为5′-前导核酶，图1A）。该核酶与蛋白质ARS具有某些功能相似性;（1）除了N-生物素化的Phe氰基甲酯外，核酶还可以使用苯丙氨酰-腺苷酸作为氨酰供体，（生物素-Phe-CME），其最初用于体外进化，（2）其关键地识别存在于otRNA的3′-末端的CCA序列和DNA碱基，和（3）其能够氨基酰化微螺旋RNA 8，19，20由otRNA的受体茎和T β C茎环组成。由于在核酶和ARS之间观察到的这些显著的相似性，我们想知道核酶是否像蛋白质ARS那样只对末端A76的2′-或3′-OH基团进行氨酰化。在早期的工作中，我们已经间接地证明了3′-末端的OH基团的必要性，证明了3′-末端二醇的高碘酸盐氧化或A76的缺失会抑制核酶的活性。确定OH基团上的特定位点也应该为otRNA的3′-末端氨酰化提供具体证据。为了探测特定的氨酰化位点，我们采取了一种方法来制备一组半合成的otRNA 10，21，其由两段otRNA片段组成（图2A），其中3′-片段RNA含有A76的脱氧类似物，即2′-或3′-脱氧腺苷（dA）。在这一组中，化学合成三种合成的19-mer RNA（图2B中的1、2和3），5′-放射性标记，然后与体外转录的56-mer otRNA片段组装，构建相应的半合成otRNA。这些分子被称为otRNA-1、otRNA-2和otRNA-3（图2A）。
The RNA-based aminoacylation system could have played a critical role in establishing an ancient genetic code. 1-4 In this system, a set of catalytic RNA molecules (ribozymes) should have been capable of aminoacylating tRNAs (or their primitive analogue) in a manner analogous to that of modern protein aminoacyl-tRNA synthetases (ARSs). 1, 4-6 We have used an in vitro evolution technique to isolate such an ARS-like ribozyme (Figure 1A) from a combinatorial pool of RNA containing 1015 unique sequences. 7 Here we report that this ribozyme is able to aminoacylate exclusively the 3′-hydroxyl (3′-OH) group of tRNA. In the modern translation system, the genetic coding event is carried out by the ARSs. 8 They generally exist in 20 different forms, each catalyzing the esterification of the specific amino acid to the 3′-terminus adenosine (A76) of the cognate tRNA isoacceptor, thereby connecting each amino acid to its corresponding tRNA’s anticodon triplet. All members of ARSs are structurally divided into two classes (I and II), 9, 10 and the selection of the esterification site is different between these classes: The class I enzymes aminoacylate the 2′-OH of A76 on tRNA, whereas class II enzymes aminoacylate the 3′-OH. 11-14 In the context of the RNA world hypothesis, 1, 15, 16 we have evolved a precursor tRNA (pre-tRNA) that specifically charges phenylalanine (Phe) to its own 3′-end. 7 This catalytic pre-tRNA is compatible with a naturally occurring endonuclease ribozyme (RNase P RNA), resulting in its fragmentation to the 5′-leader sequence and mature tRNA (otRNA, 17, 18 Figure 1B). The resultant 5′-leader segment is capable of charging Phe onto otRNA, thereby behaving as a trans-acting ARS-like ribozyme (referred to as the 5′-leader ribozyme, Figure 1A). This ribozyme exhibits some functional similarities to protein ARSs;(1) the ribozyme can use phenylalanyl-adenylate as an aminoacyl donor in addition to the N-biotinylated Phe cyanomethyl ester (biotin-Phe-CME) that was originally used for the in vitro evolution,(2) it critically recognizes the CCA sequence and discriminator base present at the 3′-terminus of otRNA, and (3) it is able to aminoacylate the minihelix RNA8, 19, 20 consisting of the acceptor stem and TΨC stem-loop of otRNA.As a result of these remarkable similarities observed between the ribozyme and ARSs, we wondered whether the ribozyme aminoacylates the 2′-or 3′-OH group of the terminal A76 exclusively, as protein ARSs do. In earlier work, 7 we have shown indirect evidence for the necessity of the OH group (s) at the 3′-end, demonstrating inhibition of the ribozyme activity by periodate oxidation of the 3′-terminal diol or deletion of A76. Determination of the specific site on either OH group should also provide concrete evidence for the 3′-end aminoacylation of otRNA. To probe the specific aminoacylation site, we took an approach to prepare a set of semi-synthetic otRNAs10, 21 consisting of two pieces of otRNA fragments (Figure 2A), where the 3′-fragment RNA contains deoxy-analogues of A76, that is, 2′-or 3′-deoxyadenosine (dA). In this set, three synthetic 19-mer RNAs (1, 2, and 3 in Figure 2B) were chemically synthesized, 5′-radiolabeled, and then assembled with an in vitro transcribed 56-mer otRNA fragment to construct the respective semi-synthetic otRNAs. These molecules were referred to as otRNA-1, otRNA-2, and otRNA-3 (Figure 2A).