Analysis of Truncated Forms of Bombyx mori Glycyl-tRNA Synthetase: Function of an N-Terminal Structure in RNA Binding

Abstract

Bombyx mori glycyl-tRNA synthetase (GRS) was expressed as the full length protein and as N-terminally and C-terminally truncated forms. The intact enzyme and forms with deletions of 12, 27, 46, and 55 N-terminal residues were expressed, purified, and characterized. All were active, having 15-25% of both pyrophosphate exchange activity and aminoacyl-tRNA synthetase activity compared to wild type enzyme. Active site titration indicated that this difference in activity was not the result of production of inactive enzyme. Sedimentation and gel filtration experiments indicate that the N-terminally deleted forms and the wild type enzyme were dimers. Deletion of 55 N-terminal residues did not result in significant effects on the Michaelis constants for ATP, glycine, or tRNA, while deletion of 108 N-terminal residues and two internal 64- and 200-residue deletions generated inactive forms. Five forms with C-terminal deletions of 24, 37, 59, 162, and 327 amino acid residues were soluble and intact but lacked detectable pyrophosphate exchange activity or aminoacyl-tRNA synthetase activity. The C-terminal sequence may be required for catalysis or to maintain a stable structure. Zone electrophoresis demonstrated the wild type enzyme bound both tRNA(Gly) and noncognate tRNA(Ala). Deletion of 55 N-terminal residues resulted in altered binding of tRNA(Gly) and eliminated binding of tRNA(Ala). The first 55 N-terminal residues are not essential for catalysis, dimerization, or substrate binding in aminoacylation but are required for RNA binding not associated with aminoacylation.

Equilibrium Unfolding of Bombyx mori Glycyl-tRNA Synthetase

“…4s, A, of the Supplemental Material) associated with changes in both the CD and fluorescence spectra. In the absence of denaturant the wild type protein had a weighted average value of S 20,w of 6.91, consistent with previous determinations of this parameter (15,47) and consistent with a dimer. At 1.5 M urea the weighted average S 20,w was 5.75.…”

“…Materials-Wild type B. mori glycyl-tRNA synthetase and a mutant lacking the first 55 N-terminal residues (N55) were expressed in E. coli BL21DE3pLysS transformed with plasmids encoding these proteins (pNADA and pNADAN55, described previously (15)). The proteins were purified by successive chromatography on Q-Sepharose, hydroxylapatite, and Sephacryl S200 (15).…”

“…The proteins were purified by successive chromatography on Q-Sepharose, hydroxylapatite, and Sephacryl S200 (15). Stock solutions of the purified proteins at 15-25 mg ml Ϫ1 in 50 mM potassium phosphate (pH 7.5), 20% (v/v) glycerol, 0.5 mM EDTA, 0.5 mM dithiothreitol were stored as small aliquots at Ϫ80°C until use.…”

“…One such element in glycyl-tRNA synthetase from Bombyx mori, Homo sapiens, Caenorhabditis elegans, and Arabadopsis thalinia is a 50-to 60-residue N-terminal structure present in a number of other eukaryotic aminoacyl-tRNA synthetases, including the glutamyl-prolyl-, histidyl-, tryptophanyl-, and methionyl-tRNA synthetases. Although the physiological function of this structure is unclear, because it is apparently not required for amino acid activation or tRNA binding, some studies suggest it may have a role in binding to polynucleotides (15,16) or in mediating interaction with other proteins (17,18). One study of human histidyl-tRNA synthetase (19) indicated that removal of this element from the N terminus resulted in a drastic reduction in enzymatic activity, although the removal of the corresponding structure in B. mori glycyl-tRNA synthetase does not have the same effect (15).…”

“…Although the physiological function of this structure is unclear, because it is apparently not required for amino acid activation or tRNA binding, some studies suggest it may have a role in binding to polynucleotides (15,16) or in mediating interaction with other proteins (17,18). One study of human histidyl-tRNA synthetase (19) indicated that removal of this element from the N terminus resulted in a drastic reduction in enzymatic activity, although the removal of the corresponding structure in B. mori glycyl-tRNA synthetase does not have the same effect (15). Studies by Raben et al (19) indicated that the structure has a high content of ␣-helix and may form a coiled-coil, similar to the N-terminal domain of the E. coli (4,20) and T. thermophilus (21,22) seryl-tRNA synthetases.…”