A Zymography technique to study amino acid activation by aminoacyl tRNA synthetases (aaRS): A broad spectrum, high-throughput tool to screen activities of aaRS and their “Urzyme” variants

Abstract

Amino acyl tRNA synthetases or aaRSs play a key role in assuring the precision of protein 13 translation. They are highly specific for their cognate amino acid and cognate tRNA 14 substrates during protein synthesis, utilizing ATP to ensure that proper assignments are made 15 between amino acid and anticodon. Specific aaRS for each amino acid are present in all cells. 16 We describe a new zymography technique to qualitatively visualize and semi-quantitatively 17 determine the amino acid activation capacity of each type of aaRS molecule by indirect 18 colorimetric detection of released pyrophosphates during the formation of aminoacyl-AMP. 19 Protein samples containing aaRS are subjected to Native PAGE, followed by incubation in 20 buffer containing cognate amino acid and ATP for sufficient time to generate pyrophosphates 21 (PPi) which are then converted to inorganic phosphates by pyrophosphatase treatment.