The control region of the mannitol ) operon of has been shown to contain five cAMP receptor protein (CRP) binding sequences, the most yet reported for any operon. A DNA fragment encompassing the entire operon regulatory region was generated by PCR, and the binding of the cAMP-CRP complex was studied. Using restrictional analysis to separate, delineate and destroy the various putative CRP binding sites, all five sites were shown to be functional for CRP binding . Four of these sites bound the cAMP-CRP complex with high affinity while the fifth site (the most distal relative to the transcriptional start site) bound the complex with lower affinity. Simultaneous binding of cAMP-CRP complexes to several of these sites was demonstrated. The results serve to identify and define five dissimilar CRP binding sites in a single operon of . A model for operon transcriptional initiation and repression complexes is presented.


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