Francklyn C, Schimmel P
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Nature. 1989 Feb 2;337(6206):478-81. doi: 10.1038/337478a0.
The genetic code is determined by both the specificity of the triplet anticodon of tRNAs for codons in mRNAs and the specificity with which tRNAs are charged with amino acids. The latter depends on interactions between tRNAs and their charging enzymes, and an advance in understanding such interactions was provided recently by the demonstration that a major determinant of the identity of alanine tRNA is located in the amino-acid acceptor helix. Multiple substitutions in many distinct parts of the molecule do not prevent aminoacylation with alanine. Substitution of the G3.U70 base pair with G3.C70 or A3.U70 in the acceptor helix prevents aminoacylation in vivo and in vitro, however, and the introduction of this base pair into tRNA(Cys) (ref. 1) or tRNA(Phe) (refs 1, 2) enables both to accept alanine. The importance of a single base pair in the acceptor helix and the results of recent footprinting experiments promoted us to investigate the possibility that a minihelix, composed only of the amino-acid acceptor-T psi C helix, could be a substrate for alanine tRNA synthetase. We show here that a synthetic hairpin minihelix can be enzymatically aminoacylated with alanine. Alanine incorporation requires a single G.U base pair, and occurs in helices that otherwise differ significantly in sequence. Aminoacylation can be achieved with only seven base pairs in the helix.
遗传密码由tRNA三联体反密码子对mRNA密码子的特异性以及tRNA负载氨基酸的特异性共同决定。后者取决于tRNA与其负载酶之间的相互作用，最近的一项研究为理解这种相互作用带来了进展，该研究表明丙氨酸tRNA身份的一个主要决定因素位于氨基酸接受螺旋中。分子许多不同部位的多个替换并不妨碍用丙氨酸进行氨基酰化。然而，在接受螺旋中将G3.U70碱基对替换为G3.C70或A3.U70会阻止体内和体外的氨基酰化，并且将这个碱基对引入tRNA（Cys）（参考文献1）或tRNA（Phe）（参考文献1、2）能使二者都接受丙氨酸。接受螺旋中单个碱基对的重要性以及最近足迹实验的结果促使我们研究仅由氨基酸接受 - TψC螺旋组成的小螺旋可能成为丙氨酸tRNA合成酶底物的可能性。我们在此表明，一种合成发夹小螺旋可以被丙氨酸酶促氨基酰化。丙氨酸掺入需要一个单一的G.U碱基对，并且发生在序列上有显著差异的螺旋中。仅用螺旋中的七个碱基对就能实现氨基酰化。
