Activation of in vitro transcription and topology of closed DNA domains

Activation of in vitro transcription of otherwise inert DNA sequences by purified yeast RNA polymerase II has been observed following in the introduction in closed DNA domains of fragments of various origin. This enhancer-like effect on the in vitro transcriptional capacity is only detected in negatively supercoiled DNA domains and is characterized for each chimaeric plasmid by the superhelical density (-?) at which a sharp transition toward activation takes place. We have analyzed the topological state (as defined by localization and evaluation of the relative occurrence of secondary structure sensitive to S1 endonuleae) of the activated closed domains as a function of the conditions that determine the transcriptional enhancer effect, i.e. superhelical density, size, and nature of the components of the domains. We observe that variations in transcriptional capacity coincide with a defined pattern of secondary structures. These observations support a cause-effect relation between topology and regulation of transcription.