Study on the reinitiation of transcription in 37 RC cells after actinomycin D inhibition. Spectrum of major RNA species resynthesized after maximal suppression by the drug.

Abstract

The reinitiation of the synthesis of major RNA species has been studied in 37 RC cells after maximal inhibition of RNA synthesis by actinomycin D (AMD). During the period of recovery from AMD, resynthesized RNA (rec-RNA) is initially composed of almost exclusively light (4-14S) heterogeneous RNA species. All normal species of RNA can be detected in the rec-RNA spectrum as early as 3 h after AMD removal. The synthesis of low molecular weight methylated RNA species increases slightly during the early period after AMD removal, while the increase of low molecular weight unmethylated species is more significant during the same period. Much of the radioactivity in the polyribosomal fraction is EDTA and puromycin sensitive. Since polysomal, puromycin-sensitive RNA is polyadenylated (as evidenced by the binding of poly-U filters), and is heterogenous in size, it belongs to the m-RNA class. The synthesis of m-RNA increases immediately after AMD removal, whereas the reinitiation of the r-RNA synthesis occurs after a lag period of about 2 h. The kinetics of recovery of the synthesis of major RNA species from AMD inhibition show a size dependency comparable to the size-related sensitivity to AMD inhibition in other cellular systems. This dependency is most clearly seen in HnRNA, the AMD sensitivity of which is measured by the length of the lag period between AMD removal and the appearance of HnRNA fractions in a sucrose density gradient. Low molecular weight HnRNA reappears first, whereas heavier fractions of HnRNA appear in the spectrum after a lag period, the length of which is in direct relation to the position of the HnRNA fraction in the gradient.