title: Homogeneous fluorescent assay for RNA polymerase.
abstract: 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.
authors.affiliation: Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA.
authors.forename: Maxim
authors.initials: M
authors.lastname: Kozlov
authors.forename: Veit
authors.initials: V
authors.lastname: Bergendahl
authors.forename: Richard
authors.initials: R
authors.lastname: Burgess
authors.forename: Alex
authors.initials: A
authors.lastname: Goldfarb
authors.forename: Arkady
authors.initials: A
authors.lastname: Mustaev
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D000617:Aminoglycosides / Q000494:pharmacology; D020650:Combinatorial Chemistry Techniques; D003374:Coumarins / Q000138:chemical synthesis*; D012321:DNA-Directed RNA Polymerases / Q000032:analysis* / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D006493:Heparin / Q000494:pharmacology; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000617:Aminoglycosides; D003374:Coumarins; D004791:Enzyme Inhibitors; C504484:adenosine-5'-tetraphosphate-delta-(4-methylumbelliferone) ester; C504482:adenosine-5'-triphosphate-gamma-(4-methylumbelliferone) ester; D006923:Hymecromone; C100307:streptolydigin; D006493:Heparin; D012321:DNA-Directed RNA Polymerases; D000241:Adenosine; D012293:Rifampin