Enhancer-activated plasmid transcription complexes contain constrained supercoiling.
It has been proposed that transcriptionally active chromatin contains totally unconstrained supercoiling. The results of recent studies have raised the possibility that this topological state is the property of highly transcribed genes. Since the transcription rate of RNA polymerase II genes can be dramatically increased by the presence of an enhancer, we have determined if the transcription complex of an enhancer-activated plasmid contains totally unconstrained supercoils. Following transfection into COS7 cells, the topology of the transcription complex DNA was determined directly by agarose gel electrophoresis. We find that an enhancer-activated plasmid transcription complex is supercoiled, and these supercoils remain following topoisomerase I treatment. Thus the transcribing complexes contain constrained supercoils, and the level of supercoiling suggests a nucleosome-like organization. However, we cannot rule out the possibility that unconstrained supercoils exist in addition to these constrained supercoils in the transcription complex in the cell.
Bonilla
P J
PJ
Molecular Biology Research Program, Henry Ford Hospital, Detroit, MI 48202.
Freytag
S O
SO
Lutter
L C
LC
D000818:Animals; D001124:Argininosuccinate Synthase / Q000235:genetics; D002460:Cell Line; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004742:Enhancer Elements, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I; D001124:Argininosuccinate Synthase