Nameki N, Asahara H, Hasegawa T
Department of Biology, Faculty of Science, Hirosaki University, Japan.
FEBS Lett. 1996 Nov 4;396(2-3):201-7. doi: 10.1016/0014-5793(96)01094-0.
In this study, we identified nucleotides that specify aminoacylation of tRNA(Thr) by Thermus thermophilus threonyl-tRNA synthetase (ThrRS) using in vitro transcripts. Mutation studies showed that the first base pair in the acceptor stem as well as the second and third positions of the anticodon are major identity elements of T. thermophilus tRNA(Thr), which are essentially the same as those of Escherichia coli tRNA(Thr). The discriminator base, U73, also contributed to the specific aminoacylation, but not the second base pair in the acceptor stem. These findings are in contrast to E. coli tRNA(Thr), where the second base pair is required for threonylation, with the discriminator base, A73, playing no roles. In addition, among several mutations at the third base pair in the acceptor stem, only the G3-U70 mutant was a poor substrate for ThrRS, suggesting that the G3-U70 wobble pair, which is the identity determinant of tRNA(Ala), acts as a negative element for ThrRS. Similar results were obtained in E. coli and yeast. Thus, this manner of rejection of tRNA(Ala) is also likely to have been retained in the threonine system throughout evolution.
在本研究中，我们利用体外转录本鉴定了嗜热栖热菌苏氨酰 - tRNA合成酶（ThrRS）对tRNA(Thr)进行氨酰化的核苷酸。突变研究表明，接受茎中的第一个碱基对以及反密码子的第二和第三个位置是嗜热栖热菌tRNA(Thr)的主要识别元件，这与大肠杆菌tRNA(Thr)的基本相同。判别碱基U73也有助于特异性氨酰化，但接受茎中的第二个碱基对则不然。这些发现与大肠杆菌tRNA(Thr)相反，在大肠杆菌中，第二个碱基对是苏氨酰化所必需的，而判别碱基A73不起作用。此外，在接受茎中第三个碱基对的几个突变中，只有G3 - U70突变体是ThrRS的不良底物，这表明作为tRNA(Ala)识别决定因素的G3 - U70摆动对作为ThrRS的负性元件起作用。在大肠杆菌和酵母中也获得了类似的结果。因此，这种排斥tRNA(Ala)的方式在整个进化过程中也可能保留在苏氨酸系统中。
