Distortion in the spacer region of P-m during activation of middle transcription of phage Mu

Transcription from the middle promoter, P-m, of phage Mu is initiated by Escherichia coli RNA polymerase holoenzyme (E sigma(70); RNAP) and the phage-encoded activator, Mor. Point mutations in the spacer region between the -10 hexamer and the Mor binding site result in changes of promoter activity in vivo. These mutations are located at the junction between a rigid T-tract and adjacent, potentially deformable G+C-rich DNA segment, suggesting that deformation of the spacer region may play a role in the transcriptional activation of P-m. This prediction was tested by using dimethyl sulfate and potassium permanganate footprinting analyses, Helical distortion involving strand separation was detected at positions -32 to -34, close to the predicted interface between Mor and RNAP. Promoter mutants in which this distortion aas not detected exhibited a lack of melting in the -12 to -1 region and reduced promoter activity in vivo. We propose that complexes containing the distortion represent stressed intermediates rather than stable open complexes and thus can be envisaged as a transition state in the kinetic pathway of P-m activation in which stored torsional energy could be used to facilitate melting around the transcription start point.