Abstract
RNA polymerase II seems to be prone to stop at intrinsic pause sites, thus introducing a further potential level of regulation. It was recently shown that RNA polymerase II was held at the P2 promoter of c-myc gene. We confirmed the presence of engaged polymerases in the murine fibroblastic Ltk- and pre-B lymphoid 70Z3 cell lines. High resolution run-on analysis and in vivo permanganate-dependent footprinting showed that this holds true for the c-fos gene in unstimulated cells where a strong block to transcription elongation was evidenced. In contrast to what was observed in the c-myc gene, an even more intense signal was observed in run-on experiments downstream to the promoter, on a c-fos oligonucleotide including position +385 where an in vitro transcription arrest site was previously mapped. Genomic footprinting of DNA from intact cells and isolated nuclei confirmed the involvement of several thymidines belonging to a T-rich stretch in a melted region which was not detected upon polymerase release. In order to observe a short abortive c-fos transcript accumulating in vivo we resorted to microinjection of c-fos templates in Xenopus oocytes where transcripts were stable.