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Nucleic Acids Research 2004 32(11):3294-3303; doi:10.1093/nar/gkh665
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Published online 18 June 2004

Nucleic Acids Research, Vol. 32 No. 11 © Oxford University Press 2004; all rights reserved

Two distinct domains of the ß subunit of Aquifex aeolicus leucyl-tRNA synthetase are involved in tRNA binding as revealed by a three-hybrid selection

Yong-Gang Zheng, Hui Wei, Chen Ling, Franck Martin1, Gilbert Eriani1 and En-Duo Wang*

State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China and 1 UPR9002, IBMC du CNRS, 15 rue René Descartes, F-67084 Strasbourg, France

* To whom correspondence should be addressed. Tel: +86 21 54921241; Fax: +86 21 54921011; Email: edwang{at}sibs.ac.cn

Received April 16, 2004; Revised May 26, 2004; Accepted June 3, 2004

The Aquifex aeolicus {alpha}ß-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the ß subunit which is believed to bind tRNALeu. A yeast three-hybrid system was constructed on the basis of the interaction of the ß subunit with its cognate tRNALeu. Then, seven mutated ß subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle-domain, which might interact with the D-hairpin of the tRNA analogous to other class Ia tRNA:synthetases complexes. The five other mutations were found in the LeuRS-specific C-terminal domain of which the folding is still unknown. tRNA affinity measurements and kinetic analyses performed on the isolated ß subunits and on the co-expressed {alpha}ß-heterodimers showed for all the mutants an effect in tRNA affinity in the ground state. In addition, an effect on the transition state of the aminoacylation reaction was observed for a 21-residues deletion mutant of the C-terminal end. These results show that the genetic approach of the three hybrid system is widely applicable and is a powerful tool for the investigation of tRNA:synthetase interactions.


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J. L. Hsu, S. B. Rho, K. M. Vannella, and S. A. Martinis
Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles
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