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Journal of Biochemistry Advance Access originally published online on January 29, 2009
Journal of Biochemistry 2009 145(5):635-641; doi:10.1093/jb/mvp020
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Molecular Recognition of Tryptophan tRNA by Tryptophanyl-tRNA Synthetase from Aeropyrum pernix K1

Wataru Tsuchiya1 and Tsunemi Hasegawa1,2,*

1Graduate School of Science and Engineering; and 2Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan

*To whom correspondence should be addressed. Tel: +81-23-628-4604, Fax:+81-23-628-4604, E-mail: hasegawa{at}sci.kj.yamagata-u.ac.jp

Received December 17, 2008; Accepted January 22, 2009


  Abstract

The identity elements of transfer RNA are the molecular basis for recognition by each cognate aminoacyl-tRNA synthetase. In the archaea system, the tryptophan tRNA identity has not been determined in detail. To investigate the molecular recognition mechanism of tryptophan tRNA by tryptophanyl-tRNA synthetase (TrpRS) from the hyperthermophilic and aerobic archaeon, Aeropyrum pernix K1, various mutant transcripts of tryptophan tRNA prepared by an in vitro transcription system were examined by overexpression of A. pernix TrpRS. Substitution of the discriminator base, A73, impaired tryptophan incorporation activity. Changing the G1–C72 base pair to other base pairs also decreased the aminoacylation activity. Substitutions of anticodon CCA revealed that the C34 and C35 mutants dramatically reduced aminoacylation with tryptophan, but the A36 mutants had the same activity as the wild type. The results indicate that the anticodon nucleotides C34, C35, discriminator base A73 and G1–C72 base pair are major recognition sites for A. pernix TrpRS.

Key Words: tRNA identity, tryptophan tRNA, tryptophanyl-tRNA synthetase, archaea, Aeropyrum pernix K1

Abbreviations: ARS, aminoacyl-tRNA synthetase; DTT, dithiothreitol; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; IPTG, isopropyl-β-D(–)-thiogalactopyranoside; OD600, optical density at 600 nm; PMSF, phenylmethylsulphonyl fluoride; TrpRS, tryptophanyl-tRNA synthetase; WT, wild type


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