Role of multiple CytR binding sites on cooperativity, competition, and induction at the Escherichia coli udp promoter

Abstract

The CytR repressor fulfills dual roles as both a repressor of transcription from promoters of the Escherichia coli CytR regulon and a co- activator in some circumstances. Transcription is repressed by a three- protein complex (cAMP receptor protein (CRP)-CytR-CRP) that is stabilized by cooperative interactions between CRP and CytR. However, cooperativity also means that CytR can recruit CRP and, by doing so, can act as a co-activator. The central role of cooperativity in regulation is highlighted by the fact that binding of the inducer, cytidine, to CytR is coupled to CytR-CRP cooperativity; this underlies the mechanism for induction. Similar interactions at the different promoters of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism but also provide differential expression of these genes. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. Recently, we showed that CytR binds specifically to multiple sites in the E. coli deoP promoter, thereby providing competition for CRP binding to CRP operator site 1 (CRP1) and CRP2 as well as cooperativity. The effect of the competition at this promoter is to negate the role of CytR in recruiting CRP. Here, we have used quantitative footprint and mobility shift analysis to investigate CRP and CytR binding to the E. coli udp promoter. Here too, we find that CytR both cooperates and competes for CRP binding. However, consistent with both the distribution of CytR recognition motifs in the sequence of the promoter and the regulation of the promoter, the competition is limited to CRP2. When cytidine binds to CytR, the effect on cooperativity is very different at the udp promoter than at the deoP2 promoter. Cooperativity with CRP at CRP1 is nearly eliminated, but the effect on CytR- CRP2 cooperativity is negligible. These results are discussed in relation to the current structural model of CytR in which the core, inducer-binding domain is tethered to the helix-turn-helix, DNA-binding domain via flexible peptide linkers.