ACTINOMYCIN-D STIMULATES THE TRANSCRIPTION OF RIBOSOMAL-RNA MINIGENESTRANSFECTED INTO MOUSE CELLS - IMPLICATIONS FOR THE IN-VIVO HYPERSENSITIVITY OF RIBOSOMAL-RNA GENE-TRANSCRIPTION

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long been known, but this effect could not be reprod uced in model systems and its molecular mechanisms remain uncertain. W e studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon tr ansient transfection into mouse cells. Low concentrations (0.01-0.08 m u g/ml) of actinomycin D caused within 1-2 h a 2-7-fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the ef fect was reversed and at 4 kb a practically complete inhibition of the formation of full-length transcripts was observed, accompanied, howev er, by an enhanced accumulation of unfinished rDNA transcripts. The de pendence of actinomycin D action on transcript length was also observe d with lacZ gene segments of different size inserted into the mouse rR NA minigenes. The transcription initiation of endogenous rRNA genes wa s also stimulated by the low doses of actinomycin D as indicated by th e enhanced synthesis of unfinished rDNA transcripts (spanning mainly t he 5' external transcribed spacer), whereas the synthesis of full-leng th transcripts was abolished. Removal of actinomycin D from the medium caused within 8-24 h a dramatic increase of the transcription from al l rRNA minigenes tested. This stimulation was also inversely related t o the size of the transcripts and varied from twofold to fivefold for the 3-4-kb transcripts to about 50-80-fold for the basic minigene tran script (395 nucleotides). The amount of endogenous aborted rDNA transc ripts was also markedly increased, but the synthesis of full-length tr anscripts was not restored even 24 h after removal of the drug. The pr esent results reproduce in a model cellular system the in vivo hyperse nsitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. Thi s effect requires only the basic rRNA gene promoter and terminator sig nals and does not depend on the G+C content of the RNA polymerase I tr anscripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and t ransfected rRNA genes. However, transcription-driven positive supercoi ling of rDNA is enhanced by actinomycin D and above a critical transcr ipt length it causes a blockage of transcription elongation and the ac cumulation of aborted rDNA transcripts.