INTEGRATION HOST FACTOR AND CYCLIC-AMP RECEPTOR PROTEIN ARE REQUIRED FOR TYRR-MEDIATED ACTIVATION OF TPL IN CITROBACTER-FREUNDII

The tpl gene of Citrobacter freundii encodes an enzyme that catalyzes the conversion of L-tyrosine to phenol, pyruvate, and ammonia, This ge ne is known to be positively regulated by TyrR, The amplitude of regul ation attributable to this transcription factor is at least 20-fold. T hree TyrR binding sites, designated boxes A, B, and C, centered at coo rdinates -272.5, -158.5, and -49.5, respectively, were identified in t he upstream region of the tpl promoter. The results of mutational expe riments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR -mediated activation of tpl required integration host factor (IHF) and the cAMP receptor protein (CRP), By DNase I footprinting, it was show n that the IHF binding site is centered at coordinate -85 and that the re are CRP binding sites centered at coordinates -220 and -250, Mutati onal alteration of the MF binding site reduced the efficiency of the t pl promoter by at least eightfold, The proposed roles of CRP and IHF a re to introduce bends into tpl promoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-l inking method, The formation of hexameric TyrR increased when tpl DNA was present. The participation of both IHF and CRP in the activation o f the tpl promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this sy stem. A model wherein TyrR, IHF, and CRP collaborate to regulate the e xpression of the tpl promoter is presented.