MULTIPLE SPECIFIC CYTR BINDING-SITES AT THE ESCHERICHIA-COLI DEOP2 PROMOTER MEDIATE BOTH COOPERATIVE AND COMPETITIVE INTERACTIONS BETWEEN CYTR AND CAMP RECEPTOR PROTEIN

Binding of cAMP receptor protein (CRP) and CytR mediates both positive and negative control of transcription from Escherichia coli deoP2. Tr anscription is activated by CRP and repressed by a multi protein CRP . CytR . CRP complex. The latter is stabilized by cooperative interacti ons between CRP and CytR. Similar interactions at the other transcript ional units of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism. A fundamenta l question in both prokaryotic and eukaryotic gene regulation is how c ombinatorial mechanisms of this sort regulate differential expression. To understand the combinatorial control mechanism at deoP2, we have u sed quantitative footprint and gel shift analysis of CRP and CytR bind ing to evaluate the distribution of ligation states. By comparison to distributions for other CytR-regulated promoters, we hope to understan d the roles of individual states in differential gene expression. The results indicate that CytR binds specifically to multiple sites at deo P2, including both the well recognized CytR site flanked by CRP1 and C RP2 and also sites coincident with CRP1 and CRP2. Binding to these mul tiple sites yields both cooperative and competitive interactions betwe en CytR and CRP. Based on these findings we propose that CytR function s as a differential modulator of CRP1 versus CRP2-mediated activation. Additional high affinity specific sites are located at deoP1 and near the middle of the 600-base pair sequence separating P1 and P2. Evalua tion of the DNA sequence requirement for specific CytR binding suggest s that a limited array of contiguous and overlapping CytR sites exists at deoP2. Similar extended arrays, but with different arrangements of overlapping CytR and CRP sites, are found at the other CytR-regulated promoters. We propose that competition and cooperativity in CytR and CRP binding are important to differential regulation of these promoter s.