Inactivation of DNA-dependent RNA polymerase from Escherichia coli by X-rays in solution

Abstract

DNA-dependent RNA polymerase from Escherichia coli (holoenzyme) was irradiated in dilute aqueous solution in the presence of air. The enzyme was inactivated as an exponential function of the dose, when the activity was assayed with calf thymus DNA as template, while with T 4 DNA as template a biphasic dose-response curve was obtained. In the initial rapid phase of inactivation 30–40 % of the activity was lost at a yield corresponding to G = 0.2. When the initiation factor σ and the core enzyme were irradiated separately, the σ factor was approximately 40 times more radiosensitive than the core enzyme. Blocking of the SH groups of the holoenzyme with cysteamine residues during irradiation gave a strong protection against the rapid initial inactivation observed with T 4 DNA as template. Cystamine incubation of the unirradiated holoenzyme gave a similar biphasic inactivation curve as irradiation. The results indicate that the rapid initial inactivation of the holoenzyme is due to preferential inactivation of the σ factor which is highly radiosensitive due to very reactive, essential sulfhydryl groups.