Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles

Seung Bae Rho,Jennifer L Hsu,Susan A Martinis,Kevin M Vannella

doi:10.1074/jbc.m601606200

Seung Bae Rho, Jennifer L Hsu + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m601606200

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Abstract

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.

Highlights

GM63107 and GM63789 and the Human Frontiers Science Program
A C-terminal Deletion Affects Splicing Complex Assembly and RNA Splicing Activity—Previously, we determined that yeast mitochondrial Leucyl-tRNA synthetase (LeuRS) and bI4 maturase simultaneously bind to the bI4 intron and stimulate splicing activity using an RNA-dependent two-hybrid assay [5]
Complementation assays have shown that a five-amino acid deletion of yeast mitochondrial LeuRS [18] and Mycobacterium tuberculosis LeuRS [6] abolish splicing activity but maintain protein synthesis

Summary

EXPERIMENTAL PROCEDURES

Materials—Oligonucleotide primers were synthesized by MWG Biotech (High Point, NC). Tritium-labeled amino acids were acquired from Amersham Biosciences. The template DNA from each PCR reaction was restriction digested by 20 units of DpnI prior to transformation of E. coli DH5␣ (Stratagene). The resin was washed once with 10 ml of HA2 buffer (10 mM Tris-HCl, 20 mM sodium Pi, pH 7.0, 500 mM NaCl, and 5% glycerol) followed by centrifugation and supernatant removal. Purified tRNAULeAuA was denatured at 80 °C for 1 min followed by the addition of 1 mM MgCl2 and quick-cooling on ice. Aminoacylation Assays—Each aminoacylation reaction contained 60 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 4 ␮M folded tRNALeu, 21 ␮M L-[3H]leucine (150 ␮Ci/ml), and 50 nM enzyme. Background signals from the cell and the buffer were subtracted from each spectrum

RESULTS

17 Ϯ 5 NMa

DISCUSSION