Abstract
Eukaryotic lysyl-tRNA synthetases (LysRS) have an N-terminal appended tRNA-interaction domain (RID) that is absent in their prokaryotic counterparts. This domain is intrinsically disordered and lacks stable structures. The disorder-to-order transition is induced by tRNA binding and has implications on folding and subsequent assembly into multi-tRNA synthetase complexes. Here, we expressed and purified RID from human LysRS (hRID) in Escherichia coli and performed a detailed mutagenesis of the appended domain. hRID was co-purified with nucleic acids during Ni-affinity purification, and cumulative mutations on critical amino acid residues abolished RNA binding. Furthermore, we identified a structural ensemble between disordered and helical structures in non-RNA-binding mutants and an equilibrium shift for wild-type into the helical conformation upon RNA binding. Since mutations that disrupted RNA binding led to an increase in non-functional soluble aggregates, a stabilized RNA-mediated structural transition of the N-terminal appended domain may have implications on the functional organization of human LysRS and multi-tRNA synthetase complexes in vivo.
Highlights
Aminoacyl-tRNA synthetases (ARSs) catalyze the aminoacylation of tRNAs, which is an essential step before protein translation can occur [1]
The previous study was based on a single-point mutagenesis study of the whole lysyl-tRNA synthetase (LysRS) protein, which have another tRNA-binding domain, so the binding affinity of the mutant hRID alone is unclear
It should be noted that hRID is devoid of the amino acids that are responsible for absorbance at 260 and 280 nm such as Phe, Tyr, Trp, and Cys (Table S1); it does not interfere with calculating the A260/A280 scores for determining nucleic acids (NAs) content
Summary
Aminoacyl-tRNA synthetases (ARSs) catalyze the aminoacylation of tRNAs, which is an essential step before protein translation can occur [1]. One of these ARSs, lysyl-tRNA synthetase (LysRS), catalyzes bond formation between tRNALys and lysine [2]. Eukaryotic LysRSs have an N-terminal appended domain that is absent in prokaryotic LysRSs [7]. This domain is known to non- interact with tRNAs in vitro [7,8], the precise role of tRNA binding on structure-functional relationships remains unclear
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