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Towards a New Aminoacylation Assay for Alanyl‐tRNA Synthetases
Author(s) -
Duplessis Kyle,
Heath Jacob,
McCarthy Ian,
Chihade Joseph
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.630.2
Subject(s) - aminoacylation , transfer rna , biochemistry , aminoacyl trna synthetase , serine , glycine , amino acyl trna synthetases , amino acid , alanine , enzyme , protein biosynthesis , chemistry , biology , rna , gene
Aminoacyl‐tRNA synthetases are crucial enzymes in protein biosynthesis, ensuring the fidelity of the genetic code by charging transfer RNAs with the appropriate amino acid. Aminoacylation is typically measured using radioactively labeled substrates, using assays that are non‐continuous, relatively expensive, and time consuming. Recently, a new assay1 was developed for tyrosyl‐tRNA synthetases in which the formation of AMP during the aminoacylation reaction is coupled to production of NADH which can be measured spectrophotometrically. To improve sensitivity, the aminoacyl‐tRNA product was regenerated through hydrolysis with a second enzymatic activity, allowing detection of aminoacylation even at low concentrations of tRNA. We have extended this assay to the alanyl‐tRNA synthetase system. Three different aminoacylation and tRNA regeneration schemes have been developed – mischarging with serine, followed by hydrolysis with the intrinsic editing domain; mischarging with glycine, followed by hydrolysis with D‐aminoacyl‐tRNA deacylase (DTD); and charging with alanine, followed by hydrolysis with a variant of DTD. Support or Funding Information Carleton College and the Towsley Foundation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .