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Published online before print November 12, 2007, 10.1261/rna.795508
RNA (2008), 14:117-126. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.
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Identification and characterization of a tRNA decoding the rare AUA codon in Haloarcula marismortui

Caroline Köhrer1, Gayathri Srinivasan1,3, Debabrata Mandal1,3, Bibekanand Mallick2, Zhumur Ghosh2, Jayprokas Chakrabarti2, and Uttam L. Rajbhandary1

1 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
2 Computational Biology Group, Theory Department, Indian Association for the Cultivation of Science, Calcutta 700032, India

Annotation of the complete genome of the extreme halophilic archaeon Haloarcula marismortui does not include a tRNA for translation of AUA, the rare codon for isoleucine. This is a situation typical for most archaeal genomes sequenced to date. Based on computational analysis, it has been proposed recently that a single intron-containing tRNA gene produces two very similar but functionally different tRNAs by means of alternative splicing; a UGG-decoding tRNATrp CCA and an AUA-decoding tRNAIle UAU. Through analysis of tRNAs from H. marismortui, we have confirmed the presence of tRNATrp CCA, but found no evidence for the presence of tRNAIle UAU. Instead, we have shown that a tRNA, currently annotated as elongator methionine tRNA and containing CAU as the anticodon, is aminoacylated with isoleucine in vivo and that this tRNA represents the missing isoleucine tRNA. Interestingly, this tRNA carries a base modification of C34 in the anticodon different from the well-known lysidine found in eubacteria, which switches the amino acid identity of the tRNA from methionine to isoleucine and its decoding specificity from AUG to AUA. The methods described in this work for the identification of individual tRNAs present in H. marismortui provide the tools necessary for experimentally confirming the presence of any tRNA in a cell and, thereby, to test computational predictions of tRNA genes.

Keywords: archaea; Haloarcula marismortui ; minor isoleucine tRNA; base modification; lysidine

Received August 24, 2007 ; accepted September 17, 2007.


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