A kinetic study on the mechanism of inhibition of RNA synthesis catalyzed by DNA-dependent RNA polymerase

The mechanism of inhibition of RNA polymerase-catalyzed synthesis of RNA by actinomycin D and the phenanthridinium derivatives ethidium bromide and 3,8-diamino-6-ethylphenanthridinium bromide (DEMB) is examined. A general kinetic equation describing the dependence of RNA synthesis on DNA template concentration is derived and distinct expressions corresponding to various possible mechanisms of inhibition are subsequently obtained by introducing into the equations assumptions as appropriate for the individual mechanisms. The fitting of the experimental results of inhibition into the resulting equations has suggested that ethidium bromide and DEMB inhibit RNA polymerase by forming an inhibitor-template complex which interferes with enzyme recognition of, and binding to, appropriate sites on the template (binding inhibition). The fitting of the dependence of the rate of RNA synthesis on the bound-inhibitor to DNA ratios to the derived kinetic expressions also allows a tentative distinction to be made as to whether ethidium bromide and DEMB interfere with RNA synthesis by a mechanism of ‘partial’ or ‘complete’ inhibition.