R-loop mapping of calf-thymus RNA polymerase II. Transcripts in vitro on restriction fragments of adenovirus 2 DNA

Nascent RNA, synthesized by calf thymus ENA polymerase II on the restriction endonuclease EcoRI fragment A and BamHI fragment B of adenovirus 2 DNA, was rehybridized to the template strand under conditions allowing transcription R-loop formation. Hybrids, visualized by electron microscopy, were found in looped and in branched configurations, the former being abundant, with an average loop number of roughly four per EcoRI fragment A and three per BamHI fragment B. Frequency distributions of transcription R-loops, synthesized on identical restriction fragments for different periods of time, showed similar patterns, thus pointing to a nonrandom type of transcription. A comparison of the frequency distribution of R-loops seen on the two overlapping restriction fragments reveals reasonable coincidence in the loop pattern in one out of the four possible fragment orientations, permitting correlation of the transcription R-loop pattern with the physical map of the adenovirus 2 DNA. Comparison of start sites tentatively mapped in vitro with the known promoters for RNA polymerase II in vivo suggests that a major transcription site in vitro is located in close proximity to, or identical with, two promoters in vivo at 16.1 and 16.5 map units.