期刊
ANALYTICAL BIOCHEMISTRY
卷 342, 期 2, 页码 206-213出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.04.022
关键词
fluorescent assay; 4-methylumbelliferone; RNA polymerase; in vitro transcription; high-throughput screening
资金
- NIGMS NIH HHS [GM28575, GM30717] Funding Source: Medline
A new method for determination of RNA polymerase (RNAP) activity is presented. The method uses nucleoside tri- and tetraphosphate derivatives carrying 4-methylumbelliferone residue at the terminal phosphate. Incorporation of such compounds in RNA by RNA polymerase is accompanied by release of di- and triphosphate derivatives of 4-methylumbelliferone. Subsequent treatment by alkaline phosphatase produces free 4-methylumbelliferone that is highly fluorescent and can be easily detected. The sensitivity of the method is higher than that reported in previous studies. The validity of the assay has been demonstrated by retrieving the RNAP inhibitors from a collection of 16,000 compounds. (c) 2005 Elsevier Inc. All rights reserved.
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