Inhibition of RNA synthesis by actinomycin D: Characteristic dose‐response of different RNA species

Abstract

A quantitative dose‐response study was made of the inhibition by actinomycin D of the synthesis of various RNA species in cultured L cells. After a suitable preincubation with various concentrations of actinomycin, synthesis was measured by a brief incorporation of radioactive uridine. The 45S precursor of ribosomal RNA, 5S ribosomal RNA, transfer RNA and various sized fractions of heterogeneous nuclear RNA were extracted from the appropriate subcellular fractions and resolved by acrylamide gel electrophoresis. Polyribosomal messenger RNA was also studied. Over the range of actinomycin concentrations employed cellular uptake of the drug is proportional to external concentration, and maximal inhibition of RNA synthesis is generally achieved within one hour after exposure. For most of the RNA species studied the dose‐response data appear to follow an exponential inactivation function with some “tailing” evident at the higher doses. Sensitivities, defined as the reciprocal of the dose necessary to reduce synthesis to 1/e of the control level, were compared and examined with respect to the molecular weight of the RNA species under consideration. Among the heterogeneous nuclear RNA's there is a good correlation between RNA size and sensitivity, such that the sensitivity per unit molecular weight is relatively constant. A similar relationship is roughly applicable to the ribosomal and transfer RNA's when taken as a group, although in this case the sensitivity per unit molecular weight is 50 to 100 fold greater. A model is proposed in which this marked difference in actinomycin sensitivity arises as a consequence of the transcription of stretches of contiguous repetitive genes by RNA polymerase molecules which attach to the DNA only at a limited number of entry points, the highest probability of attachment being at the beginning of a stretch.