Transcription regulation in vitro by anE.colipromoter containing a DNA cruciform in the ’-35‘ region

A promoter with the potential to adopt a 50 basepair (bp) cruciform spanning from -19 to -69 has been constructed in the plasmid pBR322 tetracycline resistance gene (tet) by forming an inverted repeat from '-35' sequences. Compared to a control promoter, the sequence of this cruciform promoter differs only by a 22 bp insertion between -48 and -69, upstream from the usual location of promoter sequences. The cruciform is extruded in a supercoil-dependent manner, and transcription from this promoter in vitro by RNA polymerase decreases as the negative supercoil density of the plasmid DNA increases. In contrast, transcription from the control promoter increases with negative supercoiling. Thus, DNA secondary structure in the '-35' region can affect promoter-polymerase interaction. The tet promoter cruciform also influences expression of the pBR322 beta-lactamase gene (bla). This apparently results when extrusion of the cruciform reduces the superhelicity of the plasmid molecule to a level that is below the optimum for expression from the bla promoter, illustrating one mechanism for how DNA secondary structure may effect action-at-a-distance. Transcription from both promoters in vivo does not differ from controls, suggesting that this cruciform is not generated to a significant extent intracellularly, most probably as a result of the slow kinetics of extrusion.