关键词: tRNA^Leu, 亮氨酰tRNA合成酶相互作用, 头孢菌素酰化酶, 假单孢菌, 亚基重组

Abstract:

The aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of amino acidsto their cognate tRNAs. They are specific for both amino acid and tRNA substrates to ensurethe high fidelity required by translation. Leucyl-tRNA synthetase (LeuRS) in one of aaRSsand belongs to class Ⅰ aaRSs. In order to investigate the interaction between LeuRS andtRNA^Leu, it was necessary that large amount of in vivo tRNA^Leu were isolated and various mu-tants of tRNA^Leu was obtained in vitro. In the present work Escherichia coli tRNA^Leu₁ andtRNA^Leu₂ were overproduced in Escherichia coli MT102 and isolated from the transformants,respectively. The kinetic constants of LeuRS for the tRNA^Leu were the same with previous da-ta. The leucine accepting ability of the unmodified tRNA^Leu₁ and tRNA^Leu₂ transcribed in vitrohad little difference, but only one fourth that of the modified ones. It was found avariant(LeuRS-A) of Escherichia coli LeuRS carrying a 40 residue-long duplication in its connectivepeptide 1 (CP1) domain has a 3 fold lower specificity for tRNA^Leu₁ than for tRNA^Leu₂, whereasthe native enzyme had the same specificity for their isoacceptors. By in vitro generation of aseries tRNA^Leu mutants we found that the difference on the first base pair in the acceptor stemsof the two tRNA^Leu isoacceptors lead to the different rates of leucylation catalyzed by LeuRS-Aand the flexibility of the acceptor stem of tRNA^Leu might play a key role in its recognition bythe synthetase.

Key words: tRNA^Leu, leucyl-tRNA synthetase, interaction