Multiple Specific CytR Binding Sites at the Escherichia colideoP2 Promoter Mediate Both Cooperative and Competitive Interactions between CytR and cAMP Receptor Protein

Laura T Perini,Elizabeth A Doherty,Erik Werner,Donald F Senear

doi:10.1074/jbc.271.52.33242

Laura T Perini, Elizabeth A Doherty + Show 2 more

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https://doi.org/10.1074/jbc.271.52.33242

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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 CRP.CytR.CRP 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 CytR-regulated 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.

Highlights

A key feature of the CytR regulon is that the individual cistrons are differentially expressed
Analysis of cAMP receptor protein (CRP) Binding to deoP2—Footprint titrations of CRP binding show the expected protected regions corresponding to CRP1 and CRP2 (Fig. 2)
The most significant aspect of the CytR regulon is that a complex pattern of coordinate and differential regulation of a large number of unlinked transcriptional units is provided by interactions between CRP, CytR, and the DNA sequences of the various promoters

Summary

Introduction

A key feature of the CytR regulon is that the individual cistrons are differentially expressed. 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. Others that have been investigated include tsx [7], encoding an outer membrane protein, cdd [8], encoding cytidine deaminase, udp [9] encoding uridine phosphorylase, cytr [10] encoding CytR, and most recently nupG [11] encoding a membrane nucleoside transport protein In all cases, these studies have implicated interactions of CRP and CytR with sequences located in the 80 –100 bp immediately upstream of the various transcription start sites and interactions between the proteins as the basis for positive and negative gene regulation. Given the results we report here, that until relatively recently CRP and CytR were believed to compete to bind to the same sites (cf. Ref. 4)

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