The functional and fluorescence properties of Escherichia coli RNA polymerase reacted with fluorescamine.
1. Fluorescamine (4-phenylspiro[furan-2,(3)1'-phthalan]-3,3'-dione) reacts rapidly with Escherichia coli RNA polymerase and produces a fluorescent derivative which is inactivated to an extent dependent upon reagent concentration. Excess fluorescamine is rapidly hydrolysed. Reaction is with xi-amino gruops of lysine residues in all subunits as revealed by gel electrophoresis and fluorescence scanning. 2. The extent of inactivation and fluorescence yield are diminished in the presence of added template, a finding which provides evidence for the existence of reactive and essential amino groups which can be at least partially shielded by DNA in the binary complexes. The relative decrease of fluorescence is greatest in the betabeta' subunits. Holoenzyme and core enzyme show essentially the same behavior. 3. The inactivation of activity by fluorescamine is primarily at the level of initiation. Template binding and chain propagation are less affected. 4. The enzyme derivatized by fluorescamine shows an intense fluorescence with a peak at 490 nm and an excitation maximum at 390 nm. The fluorescence lifetime is in the range of 3-8 ns and the emission is highly polarized. In reactions carried out at high ionic strength the fluorescence yield is approximately double that at low ionic strength and insensitive to the presence of template. 5. Energy transfer is observed between the derivatized enzyme as donor and ethidium bromide as acceptor in the presence of template to which both the enzyme and intercalating dye are bound. The transfer efficiency is a function of the relative concentrations and of the conditions of reaction with fluorescamine. An average transfer distance of approx. 4-5 nm has been calculated suggesting a close proximity between bound polymerase and helical regions of the template.
D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005450:Fluorescamine / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013141:Spiro Compounds / Q000494:pharmacology
D013141:Spiro Compounds; D005450:Fluorescamine; D012321:DNA-Directed RNA Polymerases