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Nucleic Acids Research, 2003, Vol. 31, No. 16 4729-4737
© 2003 Oxford University Press

A yeast knockout strain to discriminate between active and inactive tRNA molecules

Renaud Geslain, Franck Martin, Alain Camasses1 and Gilbert Eriani*

UPR 9002 SMBMR du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 Rue René Descartes, 67084 Strasbourg, France and 1 FRE2375 du CNRS, Institut de Physiologie et Chimie Biologique, 21 Rue René Descartes, 67084 Strasbourg, France

*To whom correspondence should be addressed. Tel: +33 3 88 41 70 42; Fax: +33 3 88 60 22 18; Email: g.eriani{at}ibmc.u-strasbg.fr
Present address:
Alain Camasses, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany

Here we report the construction of a yeast genetic screen designed to identify essential residues in tRNAArg. The system consists of a tRNAArg knockout strain and a set of vectors designed to rescue and select for variants of tRNAArg. By plasmid shuffling we selected inactive tRNA mutants that were further analyzed by northern blotting. The mutational analysis focused on the tRNA D and anticodon loops that contact the aminoacyl-tRNA synthetase. The anticodon triplet was excluded from the analysis because of its role in decoding the Arg codons. Most of the inactivating mutations are residues involved in tertiary interactions. These mutations had dramatic effects on tRNAArg abundance. Other inactivating mutations were located in the anticodon loop, where they did not affect transcription and aminoacylation but probably altered interaction with the translation machinery. No lethal effects were observed when residues 16, 20 and 38 were individually mutated, despite the fact that they are involved in sequence-specific interactions with the aminoacyl-tRNA synthetase. However, the steady-state levels of the aminoacylated forms of U20A and U20G were decreased by a factor of 3.5-fold in vivo. This suggests that, unlike in the Escherichia coli tRNAArg:ArgRS system where residue 20 (A) is a major identity element, in yeast this position is of limited consequence.


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