Intrinsically bent DNA flanks both sides of an RNA polymerase I transcription start site. Both regions display novel electrophoretic mobility

We have identified two intrinsically bent regions of DNA which flank the transcription start site of the rRNA gene from Physarum polycephalum. DNA fragments from both regions were analyzed by circular permutation polyacrylamide gel electrophoresis assay and computer modeling. Both types of analysis indicate that one fragment contains a relatively simple bend centered about 160 base pairs (bp) upstream of the transcription start site while the other fragment contains multiple bends, the most prominent of which is centered about 150 bp downstream of the start site. According to both gel mobilities and computer modeling we estimate that the net bending in each is about 45 degrees. These fragments were studied in detail by varying parameters of electrophoresis that are known to affect bending. Previous work indicates that anomalous mobility should decrease when temperature or ethidium bromide concentration is increased, whereas anomalous mobility should increase when polyacrylamide gel percentage is increased. The anomalous mobility of both fragments decreases as temperature is raised from 4 to 65 degrees C, although the bent structure centered at -160 bp is more temperature labile than the bend at +150 bp. Strikingly different behavior was observed for the two fragments as the polyacrylamide concentration was varied. As polyacrylamide concentrations are increased from 6 to 10%, the anomalous mobility of the bend centered at -160 bp increases while that of the bend centered at +150 bp decreases. The bend centered at +150 bp is 'straightened' at all ethidium concentrations tested. In sharp contrast and unexpectedly, the anomalous migration of the bend centered at -160 bp increases dramatically in 0.1 microgram/ml ethidium bromide. Many of the mobility differences we observe suggest that the two regions studied represent structurally distinct forms of bent DNA. The location of these strongly bent regions on either side of a RNA polymerase I transcription start site suggests important roles for such structures in chromatin structure and transcription initiation.