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Nucleic Acids Research, 2002, Vol. 30, No. 10 2103-2113
© 2002 Oxford University Press

Identification of essential domains for Escherichia coli tRNAleu aminoacylation and amino acid editing using minimalist RNA molecules

Deana C. Larkin, Amy M. Williams, Susan A. Martinis and George E. Fox*

Department of Biology and Biochemistry, 369 Science and Research Building II, University of Houston, Houston, TX 77204-5001, USA

Escherichia coli leucyl-tRNA synthetase (LeuRS) aminoacylates up to six different class II tRNAleu molecules. Each has a distinct anticodon and varied nucleotides in other regions of the tRNA. Attempts to construct a minihelix RNA that can be aminoacylated with leucine have been unsuccessful. Herein, we describe the smallest tRNAleu analog that has been aminoacylated to a significant extent to date. A series of tRNAleu analogs with various domains and combinations of domains deleted was constructed. The minimal RNA that was efficiently aminoacylated with LeuRS was one in which the anticodon stem–loop and variable arm stem–loop, but neither the D-arm nor T-arm, were deleted. Aminoacylation of this minimal RNA was abolished when the discriminator base A73 was replaced with C73 or when putative tertiary interactions between the D-loop and T-loop were disrupted, suggesting that these identity elements are still functioning in the minimized RNA. The various constructs that were significantly aminoacylated were also tested for amino acid editing by the synthetase. The anticodon and variable stem–loop domains were also dispensable for hydrolysis of the charged tRNAleu mimics. These results suggest that LeuRS may rely on identity elements in overlapping domains of the tRNA for both its aminoacylation and editing activities.

* To whom correspondence should be addressed. Tel: +1 713 743 8363; Fax: +1 713 743 8351; Email: fox{at}uh.edu


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