title: Distortion in the spacer region of Pm during activation of middle transcription of phage Mu.
abstract: Transcription from the middle promoter, Pm, 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 Pm. 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 was 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 Pm activation in which stored torsional energy could be used to facilitate melting around the transcription start point.
authors.affiliation: Department of Microbiology and Immunology, University of Tennessee, Memphis 38163, USA.
authors.forename: I
authors.initials: I
authors.lastname: Artsimovitch
authors.forename: M
authors.initials: M
authors.lastname: Kahmeyer-Gabbe
authors.forename: M M
authors.initials: MM
authors.lastname: Howe
mesh_terms: D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D018983:DNA Footprinting; D004275:DNA, Ribosomal / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli; D017895:Manganese Compounds; D008969:Molecular Sequence Data; D010087:Oxides; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins
chemical_list: D004275:DNA, Ribosomal; D017895:Manganese Compounds; C101385:Mor protein, Enterobacteria phage Mu; D010087:Oxides; D012808:Sigma Factor; D015534:Trans-Activators; D014764:Viral Proteins; C048856:permanganic acid; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I