大肠杆菌亮氨酰 - tRNA合成酶插入突变体对亮氨酰tRNA同工受体的识别

Discrimination of tRNALeu isoacceptors by the insertion mutant of Escherichia coli leucyl-tRNA synthetase.

作者信息

Li T, Li Y, Guo N, Wang E, Wang Y

机构信息

State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, China.

出版信息

Biochemistry. 1999 Jul 13;38(28):9084-8. doi: 10.1021/bi9901984.

DOI:10.1021/bi9901984

PMID:10413482

Abstract

A variant (LeuRS-A) of Escherichia coli leucyl-tRNA synthetase (LeuRS) carrying a 40-residue duplication in its connective peptide 1 (CP1) has a 3-fold lower specificity for than for, whereas wild-type LeuRS has the same specificity for these two isoacceptors. The replacement of the acceptor stem of with yields a chimeric tRNA(Leu) for which wild-type LeuRS has the same specificity as it does for the two normal isoacceptors mentioned, but for which LeuRS-A has a reduced specificity similar to that for, indicating a difference between these two acceptor stems. LeuRS-A is slightly less stable than the native enzyme. Wild-type LeuRS and LeuRS-A have almost same K(d) value for their interaction with as determined by fluorescence quenching. No difference was detected between these two proteins by CD and fluorescence spectroscopy. These results show that LeuRS-A can discriminate between the two isoacceptors of tRNA(Leu).

摘要

大肠杆菌亮氨酰 - tRNA合成酶（LeuRS）的一个变体（LeuRS - A）在其连接肽1（CP1）中携带40个残基的重复序列，对亮氨酰 - tRNA的特异性比对异亮氨酰 - tRNA的特异性低3倍，而野生型LeuRS对这两种同工受体具有相同的特异性。用异亮氨酰 - tRNA的受体茎替换亮氨酰 - tRNA的受体茎，可产生一种嵌合tRNA（Leu），对于这种嵌合tRNA，野生型LeuRS具有与上述两种正常同工受体相同的特异性，但LeuRS - A对其特异性降低，类似于对异亮氨酰 - tRNA的特异性降低，这表明这两种受体茎之间存在差异。LeuRS - A的稳定性略低于天然酶。通过荧光猝灭测定，野生型LeuRS和LeuRS - A与亮氨酰 - tRNA相互作用的K(d)值几乎相同。通过圆二色光谱和荧光光谱未检测到这两种蛋白质之间的差异。这些结果表明LeuRS - A可以区分tRNA（Leu）的两种同工受体。