STUDIES OF NUCLEOTIDE BINDING TO RIBONUCLEIC ACID POLYMERASE BY A FLUORESCENCE TECHNIQUE

Studies with fluorescence spectroscopy showed that Escherichia coli ribonucleic acid polymerase had an excitation and emission maximum at 285 and 385 mμ, respectively. The fluorescence intensity at 335 mμ was quenched by the addition of guanosine triphosphate or adenosine triphosphate. On the other hand, cytidine triphosphate and uridine triphosphate had no such effect. The dissociation constants for guanosine triphosphate-enzyme (0.12 mM) and adenosine triphosphate-enzyme (0.16 mM) complexes estimated by fluorimetric titration correlated well with the apparent Km of initiation reported previously for these two purine nucleoside triphosphates. Divalent metal was not required. Other purine nucleotides yielded dissociation constants with an order nucleoside triphosphate > nucleoside diphosphate > nucleoside monophosphate > deoxynucleoside triphosphate. Kinetic analysis of the fluorescence data suggested that guanosine triphosphate and adenosine triphosphate competed with each other for a site on the enzyme and that a specific inhibitor of initiation, rifamycin SV, also interacted with the same site (fluorophore) on the enzyme. This site is called the initiation site and it is also present on the enzyme after chromatography on phosphocellulose. The experiments reported here suggest that part of the specificity of initiation with purine nucleotides may be due to the initiation site on the ribonucleic acid polymerase. © 1969, American Chemical Society. All rights reserved.