All three elements of the lac ps promoter mediate its transcriptional response to DNA supercoiling

Abstract

The supercoiling response of four closely related promoters was examined in vitro. It was found that changes in all three elements of the lac ps promoter, i.e. the -10 sequence, the -35 sequence, and the spacer length, alter the transcriptional response to DNA supercoiling. Thus, the promoter as a whole, not just the melted region, mediates the supercoiling response. It is proposed that DNA supercoiling changes the structure of the promoter DNA to a form that can be recognized by RNA polymerase and then easily melted. All four promoter variants tested show the same qualitative response to the introduction of DNA supercoiling; that is, transcription is increased compared to relaxed DNA. However, for three of the four promoters, the rate peaked at intermediate levels of supercoiling and declined at higher superhelicities. Each mutation was found to alter both the extent of stimulation that can be achieved and the amount of superhelicity associated with maximal stimulation. The trend is that the stronger promoters are stimulated less, and this maximal stimulation occurs on templates containing fewer superhelical turns. At the level of supercoiling that may pertain in vivo, changes in superhelicity would result in considerable differential changes in the strengths of these promoters.