Direct evidence for the effect of transcription on local DNA supercoiling in vivo

Abstract

The B-to-Z structural transition of varying lengths (74 to 14 base-pairs) of (CG) tracts has been used as a superhelicity probe to examine the local topological changes induced by transcription at defined genetic loci in vivo. The local-topology reporter sequences indicate that under steady-state transcription the region upstream from the promoter experiences an increase in negative supercoiling whereas the region downstream from the terminator displays a decrease in negative superhelicity. This result provides direct in vivo evidence for the notion that the translocation of an RNA polymerase elongation complex along the double-helical DNA generates positive supercoils in front of it and negative supercoils behind it. Also, this twin-supercoiled domain model was tested inside a transcribed region where a high degree of negative supercoiling generated by the passage of each individual RNA polymerase was detected. Hence, these data indicate that the induced supercoils are confined to the vicinity of each RNA polymerase complex in a multipolymerase system.