Fine-Tuning of Alanyl-tRNA Synthetase Quality Control Alleviates Global Dysregulation of the Proteome

Abstract

One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control.

Escherichia coli alanyl-tRNA synthetase maintains proofreading activity and translational accuracy under oxidative stress

“…AlaRS was eluted using 250 mM of imidazole. The fractions containing the protein were concentrated using a 10K Amicon concentrator and dialyzed into a final buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl and 50 % glycerol (46).…”

“…The fractions containing tRNA were pooled and precipitated using 1/10 th volume of 3M sodium acetate pH 5.5 and 3X volume of ethanol at -80°C overnight. The precipitated tRNA was pelleted at 5000 xg for 25 minutes, dried and resuspended in RNase free water (46). The concentration of active tRNA was determined by aminoacylation reactions.…”