Multiple Specific CytR Binding Sites at the Escherichia coli deoP2 Promoter Mediate Both Cooperative and Competitive Interactions between CytR and cAMP Receptor Protein

Abstract

Binding of cAMP receptor protein (CRP) and CytR mediates both positive and negative control of transcription from Escherichia coli deoP2. Transcription is activated by CRP and repressed by a multi-protein CRP⅐CytR⅐CRP complex. The latter is stabilized by cooperative interactions between CRP and CytR. Similar interactions at the other transcriptional units of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. To understand the combinatorial control mechanism at deoP2, we have used quantitative footprint and gel shift analysis of CRP and CytR binding to evaluate the distribution of ligation states. By comparison to distributions for other CytRregulated promoters, we hope to understand the roles of individual states in differential gene expression. The results indicate that CytR binds specifically to multiple sites at deoP2, including both the well recognized CytR site flanked by CRP1 and CRP2 and also sites coincident with CRP1 and CRP2. Binding to these multiple sites yields both cooperative and competitive interactions between CytR and CRP. Based on these findings we propose that CytR functions 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. Evaluation of the DNA sequence requirement for specific CytR binding suggests 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 promoters.In Escherichia coli, the enzymes and transport proteins required for nucleoside catabolism and recycling are encoded by genes belonging to the CytR regulon. This gene family consists of at least nine unlinked transcriptional units (for review, see Ref. 1). Expression of these transcriptional units is coordinately regulated by the interplay of two transcriptional regulatory proteins, CRP 1 (also referred to as CAP) and the CytR repressor. Transcription is activated in response to intracellular cAMP levels by CRP, repressed by CytR, and induced by cytidine. A few of the transcriptional units are also separately regulated by a second repressor, DeoR (2-4), via an independent mechanism.A key feature of the CytR regulon is that the individual cistrons are differentially expressed. Extents of activation, repression, and induction all vary among the different transcription units (cf. Ref. 5). This is achieved by nesting levels of local repression, mediated by DeoR and CytR, on a more global regulation mediated by CRP. This illustrates a process, common to both E. coli and higher order eukaryotes, in ...

CpxR/OmpR Interplay Regulates Curli Gene Expression in Response to Osmolarity inEscherichia coli

“…We suggest that CpxR, via its specific binding sites arranged over an extended region of DNA, functions as a modulator of OmpR activation. The occurrence of such multiple repressor binding sites has been described for CytR in the deoP2 promoter (41). The present study reveals the complexity of the control exerted on the csgD expression.…”

Allosteric Mechanism of Induction of CytR-regulated Gene Expression

“…CytR was the titrating ligand. DNase I footprint titrations were conducted as described (12) in the bis-Tris (pH 7) cytidine binding buffer described above. CRP and cAMP were added to final concentrations of 0.10 M (as dimer) and 100 M, respectively.…”

“…These interactions presumably function to direct both a multistage activation of transcription, using both Class I and Class II CRP mechanisms (14) and also a similar multistage repression mediated by CytR. We have proposed that this might be a general feature of CytR-mediated gene regulation (12).…”

“…The cooperativity to which cytidine binding is linked appears to be complementary pair wise in nature. This follows from the observation that the free energy change characterizing cooperativity in the three protein complex, CRP⅐CytR⅐CRP bound to DNA, is equal to the sum of free energy changes characterizing pair wise cooperativity between CytR and CRP bound either to CRP1 or to CRP2 (12). If cooperativity in the three-protein repression complex is pairwise, then it is easy to envision that the two subunits of the dimer might react independently to cytidine binding.…”

“…Recently, we showed that CytR binds to multiple operators at one CytR regulated promoter, deoP2 (12). CytR binding to the operator responsible for repression interacts cooperatively with CRP binding to flanking CRP sites, CRP1 and CRP2.…”