Distortion in the spacer region of Pm during activation of middle transcription of phage Mu.
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.
Artsimovitch
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Department of Microbiology and Immunology, University of Tennessee, Memphis 38163, USA.
Kahmeyer-Gabbe
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Howe
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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
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