Functional Dissection of the Eukaryotic-specific tRNA-interacting Factor of Lysyl-tRNA Synthetase

Abstract

In the cytoplasm of higher eukaryotic cells, aminoacyl-tRNA synthetases (aaRSs) have polypeptide chain extensions appended to conventional prokaryotic-like synthetase domains. The supplementary domains, refered to as tRNA-interacting factors (tIFs), provide the core synthetases with potent tRNA-binding capacities, a functional requirement related to the low concentration of free tRNA prevailing in the cytoplasm of eukaryotic cells. Lysyl-tRNA synthetase is a component of the multi-tRNA synthetase complex. It exhibits a lysine-rich N-terminal polypeptide extension that increases its catalytic efficiency. The functional characterization of this new type of tRNA-interacting factor has been conducted. Here we describe the systematic substitution of the 13 lysine or arginine residues located within the general RNA-binding domain of hamster LysRS made of 70 residues. Our data show that three lysine and one arginine residues are major building blocks of the tRNAbinding site. Their mutation into alanine led to a reduced affinity for tRNA 3 Lys or minimalized tRNA mimicking the acceptor-T⌿C stem-loop of tRNA 3Lys and a decrease in catalytic efficiency similar to that observed after a complete deletion of the N-terminal domain. Moreover, covalent continuity between the tRNA-binding and core domain is a prerequisite for providing LysRS with a tRNA binding capacity. Thus, our results suggest that the ability of LysRS to promote tRNA Lys networking during translation or to convey tRNA 3 Lys into the human immunodeficiency virus type 1 viral particles rests on the addition in evolution of this tRNAinteracting factor.

Viral Hijacking of Mitochondrial Lysyl-tRNA Synthetase

“…Indeed, most of the other mitochondrial species of aminoacyl-tRNA synthetases are encoded by genes that are distinct from their cytoplasmic counterparts (2). Interestingly, mito-LysRS shares with cyto-LysRS the eukaryote-specific tRNA-binding domain, referred to as a tIF (tRNA-interacting factor), that provides eukaryotic LysRS with potent tRNAbinding properties compared with the bacterial enzyme (9,10). Mutational analysis identified residues K19, K23, R24, and K27 from the N-terminal polypeptide extension of cyto-LysRS as important for the binding of the tRNA molecule (10).…”

“…Interestingly, mito-LysRS shares with cyto-LysRS the eukaryote-specific tRNA-binding domain, referred to as a tIF (tRNA-interacting factor), that provides eukaryotic LysRS with potent tRNAbinding properties compared with the bacterial enzyme (9,10). Mutational analysis identified residues K19, K23, R24, and K27 from the N-terminal polypeptide extension of cyto-LysRS as important for the binding of the tRNA molecule (10). The amino acid residues K23, R24, and K27 are common to mitoand cyto-LysRS (Fig.…”

“…Hamster and human LysRS share 43% identical residues with bacterial LysRS (1,26) but display N-terminal polypeptide extensions that provide the core synthetases with potent tRNA-binding capacities (9,10). This N-terminal tRNA-binding domain is required for triggering tRNA Lys packaging into human immunodeficiency virus type I viral particles (3).…”

Aminoacyl-tRNA synthetase complexes: beyond translation

“…However, we cannot exclude the alternative possibility -that they were inserted into the ARS structure to augment the catalytic efficiency of the enzymes. Some ARSs also contain cis-acting motifs that are structurally unrelated to the common domains found in Arc1p, p43 and Trbp111 (Cahuzac et al, 2000;Frugier et al, 2000;Kaminska et al, 2001;Francin et al, 2002;Francin and Mirande, 2003) (Figs 2, 4). They usually form amphiphatic helices in which one side of the helix displays an array of basic residues for interaction with tRNAs (Fig.…”

“…Interestingly, many ARSs also have cis-acting domains that appear to facilitate the recruitment of tRNAs to their catalytic sites (Cahuzac et al, 2000;Frugier et al, 2000;Francin et al, 2002;Francin and Mirande, 2003). For example, the C-terminus of E. coli MRS (Morales et al, 1999), and the N-terminus of the E. coli FRS β-subunit (Simos et al, 1996) (Fig.…”