A Viable Amino Acid Editing Activity in the Leucyl-tRNA Synthetase CP1-splicing Domain Is Not Required in the Yeast Mitochondria

Kiranmai Poruri,Susan A Martinis,Michal T Boniecki,Vrajesh A Karkhanis

doi:10.1074/jbc.m607406200

Kiranmai Poruri, Susan A Martinis + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m607406200

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Abstract

Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase.

Highlights

Linus Pauling had originally predicted that the rate of misactivation of isosteric substrates that differed by one methyl group would be as high as 1 in 5 [5]
Yeast Mitochondrial LeuRS Is Active in Post-transfer Editing—The CP1 domain of LeuRS contains a threonine-rich region that comprises a portion of the editing active site [13, 15, 31, 33, 46]
This included a double mutation of two neighboring threonines (Thr-263 and -264) to valine (TT/VV), which abolishes post-transfer editing activity of E. coli LeuRS [34]

Summary

Introduction

Linus Pauling had originally predicted that the rate of misactivation of isosteric substrates that differed by one methyl group (for example, alanine and glycine or isoleucine and valine) would be as high as 1 in 5 [5]. Hydrolysis assays were carried out using 60 mM Tris, pH 7.5, 10 mM MgCl2, 150 mM KCl, and 300 nM mischarged yeast mitochondrial tRNALeu. As indicated above, including KCl increases the post-transfer editing activity of yeast mitochondrial LeuRS.2 The reaction was initiated by the addition of 50 nM enzyme and carried out at room temperature [15].

Results

Conclusion