Static DNA Bending and Protein Interactions Within thePasteurella haemolyticaLeukotoxin Promoter Region: Development of an Activation Model for Leukotoxin Transcriptional Control

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Abstract
In this study, we define cis-acting elements involved in regulation of the Pasteurella haemolytica leukotoxin promoter. In place of a canonical promoter −35 sequence, the leukotoxin promoter contains four adenine-rich repeats of sequence CA6(C/T)A, phased at approximately 10-base intervals. DNA fragments containing these repeats exhibit retarded mobility in polyacrylamide gels and permitted identification of a static DNA bend in the promoter −70 region. Deletion of the static DNA bend caused a two-fold reduction of leukotoxin transcription in Escherichia coli, suggesting that it is involved in promoter regulation. Three putative upstream activator sites (UAS), similar to those that bind the NifA activator in Klebsiella pneumoniae, are found 130 bp upstream from the transcription start site and are protected from DNase I cleavage by a P. haemolytica-specific factor. The promoter region also binds the DNA bending protein, the integration host factor (IHF), although IHF mutations do not affect its expression in E. coli. The arrangement of these elements suggests that leukotoxin expression is activated by a factor that interacts with the UAS and regulates transcription initiation at a distance via DNA looping. Activation and DNA bending may also influence a second, divergent promoter that lies 340 bp upstream from the leukotoxin start site.