Homogeneous fluorescent assay for RNA polymerase.
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.
Kozlov
Maxim
M
Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA.
Bergendahl
Veit
V
Burgess
Richard
R
Goldfarb
Alex
A
Mustaev
Arkady
A
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
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