| Assays for transfer RNA-dependent amino acid biosynthesis. | |
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MedLine Citation:
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PMID: 18241795 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Kelly Sheppard; Pierre-Marie Akochy; Dieter Söll |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S. |
Journal Detail:
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Title: Methods (San Diego, Calif.) Volume: 44 ISSN: 1046-2023 ISO Abbreviation: Methods Publication Date: 2008 Feb |
Date Detail:
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Created Date: 2008-02-04 Completed Date: 2008-05-05 Revised Date: 2012-08-01 |
Medline Journal Info:
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Nlm Unique ID: 9426302 Medline TA: Methods Country: United States |
Other Details:
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Languages: eng Pagination: 139-45 Citation Subset: IM |
Affiliation:
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Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acids
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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 | |
ID/Acronym/Agency:
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GM22854/GM/NIGMS NIH HHS; R01 GM022854-32/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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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 3.1.30.1/Single-Strand Specific DNA and RNA Endonucleases |
| Comments/Corrections | |
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