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

摘要

ABSTRACTIn this study, we definecis-acting elements involved in regulation of thePasteurella haemolyticaleukotoxin 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 inEscherichia coli, suggesting that it is involved in promoter regulation. Three putative upstream activator sites (UAS), similar to those that bind the NifA activator inKlebsiella pneumoniae, are found 130 bp upstream from the transcription start site and are protected from DNase I cleavage by aP. 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 inE. 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 distanceviaDNA looping. Activation and DNA bending may also influence a second, divergent promoter that lies 340 bp upstream from the leukotoxin start s