Article ID Journal Published Year Pages File Type
1994386 Methods 2008 7 Pages PDF
Abstract

Selenocysteinyl-tRNASec, cysteinyl-tRNACys, glutaminyl-tRNAGln, and asparaginyl-tRNAAsn in many organisms are formed in an indirect pathway in which a non-cognate amino acid is first attached to the tRNA. This non-cognate amino acid is then converted to the cognate amino acid by a tRNA-dependent modifying enzyme. The in vitro characterization of these modifying enzymes is challenging due to the fact the substrate, aminoacyl-tRNA, is labile and requires a prior enzymatic step to be synthesized. The need to separate product aa-tRNA from unreacted substrate is typically a labor- and time-intensive task; this adds another impediment in the investigation of these enzymes. Here, we review four different approaches for studying these tRNA-dependent amino acid modifications. In addition, we describe in detail a [32P]/nuclease P1 assay for glutaminyl-tRNAGln and asparaginyl-tRNAAsn formation which is sensitive, enables monitoring of the aminoacyl state of the tRNA, and is less time consuming than some of the other techniques. This [32P]/nuclease P1 method should be adaptable to studying tRNA-dependent selenocysteine and cysteine synthesis.

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Life Sciences Biochemistry, Genetics and Molecular Biology Biochemistry
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