Identification of essential domains for Escherichia coli tRNA(leu) aminoacylation and amino acid editing using minimalist RNA molecules.

Abstract:

:Escherichia coli leucyl-tRNA synthetase (LeuRS) aminoacylates up to six different class II tRNA(leu) 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 tRNA(leu) analog that has been aminoacylated to a significant extent to date. A series of tRNA(leu) 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 tRNA(leu) mimics. These results suggest that LeuRS may rely on identity elements in overlapping domains of the tRNA for both its aminoacylation and editing activities.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Larkin DC,Williams AM,Martinis SA,Fox GE

doi

10.1093/nar/30.10.2103

keywords:

subject

Has Abstract

pub_date

2002-05-15 00:00:00

pages

2103-13

issue

10

eissn

0305-1048

issn

1362-4962

journal_volume

30

pub_type

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