RNA polymerase II allows unwinding and rewinding of the DNA and thus maintains a constant length of the transcription bubble

Abstract

Ternary complexes of RNA-DNA-RNA polymerase II, originating from the in vivo transcriptionally active SV40 minichromosomes, can be detected and analyzed by a method previously developed (Choder, M., Bratosin, S., and Aloni, Y. (1984) EMBO J. 3, 2929-2936). Using this method, we compared the electrophoretic mobilities of SV40 ternary complexes with those of SV40 RNA-DNA complexes obtained after the removal of the polymerases. Independent of the in vitro elongation of the nascent RNA, topoisomers of ternary complexes including supercoiled DNA (DNA I) and relaxed DNA (DNA II) electrophoresed on agarose gels as sharp bands. In addition, the polymerase protected 18-22 nucleotides of the nascent RNA from RNase A and T1 digestion. These results demonstrate the constancy of the length of the unwound region in the transcription bubble. In contrast, following the removal of the polymerases topoisomers of RNA-DNA I complexes migrated on agarose gels as a smear between the sites of DNA I and DNA II. RNA with an average length of 120 nucleotides formed hybrid with the DNA and was RNase A- and T1-resistant. The observations that long hybrid formation is prevented as long as the polymerases are present, and the maintenance of the constant length of the transcription bubble during transcription elongation, suggest that during elongation the enzyme that actively unwinds the template allows the synchronous displacement of the nascent transcript and rewinding of the template.