STUDIES ON PHAGE-T7 DNA-DEPENDENT RNA-POLYMERASE USING GTP ANALOGS - AFFINITY MODIFICATION BY FLUORESCENT LABELING AND STUDIES OF INTERACTION WITH TEMPLATE

The interaction of bacteriophage T7 DNA-dependent RNA polymerase with three GTP analogs containing substituted naphthalenesulfonic acid resi dues has been studied Under specific conditions the analogs are irreve rsible covalent enzyme modifiers, the modified enzyme being fluorescen tly labeled and lacking catalytic activity. Modification with an analo g bearing a reactive 5'-O-(2-bromoethyl)phosphonate group blocked the ability of the enzyme to bind to polynucleotide templates. Modificatio n of the enzyme by two other analogs with azide reactive groups did no t change its template affinity, but resulted in enhanced fluorescence of the modified enzyme when bound to polynucleotides. These two fluore scent labels are suitable for fluorimetric studies of enzyme-promotor interactions.