Kiga D, Sakamoto K, Sato S, Hirao I, Yokoyama S
Yokoyama CytoLogic Project, ERATO, JST c/o RIKEN, Hirosawa, Wako-shi, Saitama, Japan.
Eur J Biochem. 2001 Dec;268(23):6207-13. doi: 10.1046/j.0014-2956.2001.02570.x.
Cytidine in the anticodon second position (position 35) and G or U in position 36 of tRNAArg are required for aminoacylation by arginyl-tRNA synthetase (ArgRS) from Escherichia coli. Nevertheless, an arginine-accepting amber suppressor tRNA with a CUA anticodon (FTOR1Delta26) exhibits suppression activity in vivo [McClain, W.H. & Foss, K. (1988) Science, 241, 1804-1807]. By an in vitro kinetic study with mutagenized tRNAs, we showed that the arginylation of FTOR1Delta26 involves C34 and U35, and that U35 can be replaced by G without affecting the activity. Thus, the positioning of the essential nucleotides for the arginylation is shifted to the 5' side, by one residue, in the suppressor tRNAArg. We found that the shifted positioning does not depend on the tRNA sequence outside the anticodon. Furthermore, by a genetic method, we isolated a mutant ArgRS that aminoacylates FTOR1Delta26 more efficiently than the wild-type ArgRS. The isolated mutant has mutations at two nonsurface amino-acid residues that interact with each other near the anticodon-binding site.
来自大肠杆菌的精氨酰 - tRNA合成酶（ArgRS）进行氨酰化反应时，tRNAArg反密码子第二位（第35位）的胞苷以及第36位的G或U是必需的。然而，一种带有CUA反密码子的精氨酸接受型琥珀抑制tRNA（FTOR1Delta26）在体内表现出抑制活性[麦克莱恩，W.H. & 福斯，K.（1988年）《科学》，241卷，1804 - 1807页]。通过对诱变tRNA进行体外动力学研究，我们发现FTOR1Delta26的精氨酰化涉及C34和U35，并且U35可以被G取代而不影响活性。因此，在抑制tRNAArg中，精氨酰化必需核苷酸的位置向5'端移动了一个残基。我们发现这种位置的移动不依赖于反密码子之外的tRNA序列。此外，通过遗传学方法，我们分离出了一种突变型ArgRS，它比野生型ArgRS更有效地对FTOR1Delta26进行氨酰化。分离出的突变体在靠近反密码子结合位点处两个相互作用的非表面氨基酸残基上发生了突变。
