Spectroscopic techniques for study of phosphodiester bond formation by Escherichia coli RNA polymerase.
Nucleotides containing the fluorophore 1-aminonaphthalene-5-sulfonate attached to the gamma phosphoryl group via a phosphoamidate bond are excellent substrates for Escherichia coli DNA-dependent RNA polymerase. Cleavage of the alpha-beta-phosphoryl bond produces significant changes in both absorption and fluorescence spectra. These alterations provide a sensitive and precise means for continuous monitoring of transcription. Under appropriate conditions one can detect the utilization of less than 1 nmol of nucleotide. Since the spectroscopic techniques measure nucleotide utilization they can be used in conjunction with measurements of incorporation of radiolabeled precursor such as [3H]UTP into acid-insoluble material to determine whether significant amounts of acid-soluble oligonucleotides are formed.
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009282:Naphthalenesulfonates; D009943:Organophosphorus Compounds; D010727:Phosphoric Diester Hydrolases; D012329:RNA, Bacterial / Q000096:biosynthesis; D013050:Spectrometry, Fluorescence
D009282:Naphthalenesulfonates; D009943:Organophosphorus Compounds; D012329:RNA, Bacterial; D012321:DNA-Directed RNA Polymerases; D010727:Phosphoric Diester Hydrolases