Document Detail

Assays for transfer RNA-dependent amino acid biosynthesis.
MedLine Citation:
PMID:  18241795     Owner:  NLM     Status:  MEDLINE    
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.
Kelly Sheppard; Pierre-Marie Akochy; Dieter Söll
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Methods (San Diego, Calif.)     Volume:  44     ISSN:  1046-2023     ISO Abbreviation:  Methods     Publication Date:  2008 Feb 
Date Detail:
Created Date:  2008-02-04     Completed Date:  2008-05-05     Revised Date:  2014-01-08    
Medline Journal Info:
Nlm Unique ID:  9426302     Medline TA:  Methods     Country:  United States    
Other Details:
Languages:  eng     Pagination:  139-45     Citation Subset:  IM    
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MeSH Terms
Amino Acids / biosynthesis*
Carbon Radioisotopes
Chromatography, High Pressure Liquid
Chromatography, Thin Layer
Metabolic Networks and Pathways
Phosphorus Radioisotopes
RNA, Transfer / metabolism*
RNA, Transfer, Amino Acid-Specific / biosynthesis
RNA, Transfer, Amino Acyl / biosynthesis
RNA, Transfer, Asn / biosynthesis
Single-Strand Specific DNA and RNA Endonucleases / metabolism
Grant Support
Reg. No./Substance:
0/Amino Acids; 0/Carbon Radioisotopes; 0/Phosphorus Radioisotopes; 0/RNA, Transfer, Amino Acid-Specific; 0/RNA, Transfer, Amino Acyl; 0/RNA, Transfer, Asn; 0/tRNA, selenocysteine-; 9014-25-9/RNA, Transfer; EC Specific DNA and RNA Endonucleases

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