Assays for transfer RNA-dependent amino acid biosynthesis.
Selenocysteinyl-tRNA(Sec), cysteinyl-tRNA(Cys), glutaminyl-tRNA(Gln), and asparaginyl-tRNA(Asn) 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-tRNA(Gln) and asparaginyl-tRNA(Asn) 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.
Sheppard
Kelly
K
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA.
Akochy
Pierre-Marie
PM
Söll
Dieter
D
D000596:Amino Acids / Q000096:biosynthesis*; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D053858:Metabolic Networks and Pathways; D010761:Phosphorus Radioisotopes; D012343:RNA, Transfer / Q000378:metabolism*; D012345:RNA, Transfer, Amino Acid-Specific / Q000096:biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis; D012348:RNA, Transfer, Asn / Q000096:biosynthesis; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D000596:Amino Acids; D002250:Carbon Radioisotopes; D010761:Phosphorus Radioisotopes; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; D012348:RNA, Transfer, Asn; C036928:tRNA, selenocysteine-; D012343:RNA, Transfer; D015719:Single-Strand Specific DNA and RNA Endonucleases