ACTIVATION OF PAUSING RNA-POLYMERASES BY NUCLEAR RUN-ON EXPERIMENTS

The nuclear run-on transcription assay is the only approach to measure the transcriptional activity of a given gene in its genuine structura l and regulatory cellular context. However, serious problems in the in terpretation of results can arise from the artificial activation of pa used RNA polymerases during the transcription reaction, leading to fal se results with regard to the level and mode of gene regulation in viv o. We have used the example of the human proto-oncogene c-myc, which h as previously been reported to be regulated by premature termination o f transcription, to describe the problems and pitfalls in the interpre tation of nuclear run-on experiments. We show here that activation of paused, elongation-incompetent polymerases in nuclear run-on experimen ts produces a strong transcription signal on c-myc exon 1 in cells whi ch do not express c-myc steady-state RNA. (C) 1994 Academic Press, Inc .