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

Open AccessPublished:December 27, 1996DOI:https://doi.org/10.1074/jbc.271.52.33242
      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.

      INTRODUCTION

      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.
      • Hammer-Jespersen K.
      ). Expression of these transcriptional units is coordinately regulated by the interplay of two transcriptional regulatory proteins, CRP
      The abbreviations used are: CRP
      E. coli cAMP receptor protein; bp, base pair(s); bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol
      RNAP
      E. coli RNA polymerase
      CytR
      E. coli cytidine repressor protein
      CRP1 and CRP2
      CRP operator sites 1 and 2, respectively
      P1 and P2
      deo operon promoters 1 and 2, respectively.
      (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 (
      • Valentin-Hansen P.
      • Albrechtsen B.
      • Love L.J.
      ,
      • Bremer E.
      • Gerlach P.
      • Middendorf A.
      ,
      • Munch-Petersen A.
      • Jensen N.
      ), 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.
      • Martinussen J.
      • Mollegaard N.E.
      • Holst B.
      • Douthwaite S.R.
      • Valentin-Hansen P.
      ). 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 which complex patterns of expression are controlled using a small number of regulatory proteins. For example regulation of cell growth and differentiation often combines tissue-specific or developmental stage-specific factors with more global control elements. The mechanism of such broad regulatory programs is a fundamental issue in gene regulation. Presumably combinatorial mechanisms that rely on different local features of different genes are involved.
      Several of the CytR-regulated operons have been investigated at the molecular level. The promoter deoP2 of the tetracistronic operon that directs the synthesis of purine and pyrimidine phosphorylases and enzymes required for sugar utilization has generated the greatest interest (
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ). Others that have been investigated include tsx (
      • Gerlach P.
      • Soogard-Andersen L.
      • Pedersen H.
      • Martinussen J.
      • Valentin-Hansen P.
      ), encoding an outer membrane protein, cdd (
      • Holst B.
      • Sogaard A.L.
      • Pedersen H.
      • Valentin-Hansen P.
      ), encoding cytidine deaminase, udp (
      • Brikun I.
      • Suziedelis K.
      • Stemmann O.
      • Zhong R.
      • Alikhanian L.
      • Linkova E.
      • Mironov A.
      • Berg D.E.
      ) encoding uridine phosphorylase, cytr (
      • Pedersen H.
      • Sogaard-Andersen L.
      • Holst B.
      • Gerlach P.
      • Bremer E.
      • Valentin-Hansen P.
      ) encoding CytR, and most recently nupG (
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ) 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. Thus, differential and coordinate gene regulation must depend on different dispositions of CRP and CytR binding sites, different protein binding affinities, and/or different levels of site-site interaction or cooperativity.
      Most CytR-regulated promoters contain tandem CRP sites. CRP1 (at about −41.5 bp from transcription start sites) and CRP2 (at about −92.5 bp) appear to be class II and class I sites, respectively (
      • Ushida C.
      • Aiba H.
      ,
      • Ebright R.H.
      ). The significance of these classifications is the suggestion that they direct different kinetic mechanisms of activation. At class I sites, CRP is proposed to increase the apparent affinity of RNAP for the promoter, whereas at class II sites, CRP is proposed to increase the rate of formation of the open transcription complex. At deoP2, CRP1 alone, but not CRP2 alone, substantially activates transcription (
      • Martinussen J.
      • Mollegaard N.E.
      • Holst B.
      • Douthwaite S.R.
      • Valentin-Hansen P.
      ). CRP1 has been proposed to substitute for the lack of a −35 consensus promoter sequence (
      • Valentin-Hansen P.
      • Holst B.
      • Sogaard A.L.
      • Martinussen J.
      • Nesvera J.
      • Douthwaite S.R.
      ), whereas CRP2 is proposed to be necessary only for CytR to bind. However, in the absence of kinetic studies on any of these promoters there is little direct evidence to suggest which kinetic mechanism of activation is involved under any condition. The highest levels of expression are achieved with both sites functional, suggesting some synergy in activation (
      • Sogaard-Andersen L.
      • Martinussen J.
      • Mollegaard N.E.
      • Douthwaite S.R.
      • Valentin-Hansen P.
      ).
      The tandem CRP sites in different promoters are separated by DNA of variable length and sequence. Based on footprinting analysis of purified CytR binding to deoP2 and other promoters, this intervening sequence is now thought to contain the CytR binding site (
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ,
      • Holst B.
      • Sogaard A.L.
      • Pedersen H.
      • Valentin-Hansen P.
      ,
      • Sogaard-Andersen L.
      • Pedersen H.
      • Holst B.
      • Valentin-Hansen P.
      ). A putative recognition motif has been identified (
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ,
      • Rasmussen P.B.
      • Sogaard A.L.
      • Valentin-Hansen P.
      ), and pairs of such motifs, arranged as either direct or inverted repeats, have been implicated as the CytR operator (
      • Brikun I.
      • Suziedelis K.
      • Stemmann O.
      • Zhong R.
      • Alikhanian L.
      • Linkova E.
      • Mironov A.
      • Berg D.E.
      ,
      • Rasmussen P.B.
      • Sogaard A.L.
      • Valentin-Hansen P.
      ). It is ironic, 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.
      • Munch-Petersen A.
      • Jensen N.
      ).
      The mechanism of CytR-mediated repression is indirect. CytR has no effect on basal level transcription but instead requires CRP binding to mediate repression (
      • Sogaard-Andersen L.
      • Pedersen H.
      • Holst B.
      • Valentin-Hansen P.
      ,
      • Sogaard-Andersen L.
      • Valentin-Hansen P.
      ). Of course, this necessarily means that CytR functions only under conditions of CRP-dependent activation. Under these circumstances CytR typically does not completely reduce the activated level of expression to the basal level (
      • Martinussen J.
      • Mollegaard N.E.
      • Holst B.
      • Douthwaite S.R.
      • Valentin-Hansen P.
      ). Despite this role of CytR as a functional antagonist of CRP, the two proteins interact cooperatively, resulting in substantially increased CytR binding affinity when CRP is present (
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ). The role of cooperativity is widely thought to be to recruit CytR at otherwise sub-saturating concentrations. The crucial role that cooperativity plays in repression is highlighted by the fact that when CytR binds cytidine, induction occurs as a result of the loss of cooperativity and despite no effect of cytidine binding to CytR on intrinsic binding of CytR to DNA (
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ).
      C. S. Barbier, S. A. Short, and D. F. Senear, submitted for publication.
      The complexity of these regulatory properties has generated confusion about the nature of key molecular interactions. The most perplexing questions involve the CRP-CytR cooperativity. For example, heterologous cooperativity has been reported to require both CRP sites, from which a lack of pairwise interactions is inferred (
      • Sogaard-Andersen L.
      • Mollegaard N.E.
      • Douthwaite S.R.
      • Valentin-Hansen P.
      ). Yet, regulation of cytRP, in which there is only one CRP site, also depends on cooperative CytR and CRP binding (
      • Pedersen H.
      • Sogaard-Andersen L.
      • Holst B.
      • Gerlach P.
      • Bremer E.
      • Valentin-Hansen P.
      ). In addition, it was recently observed (
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ,
      • Perini L.
      • Doherty E.A.
      • Senear D.F.
      ) that the apparent cooperativity, when assessed by the effect of saturating CRP concentration on CytR affinity, is substantially greater than when cooperativity is assessed by the effect of saturating CytR concentration on CRP binding. This “one-way stimulation” (
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ) represents an apparent conflict with the laws of thermodynamics that still awaits a molecular explanation.
      Several lines of evidence implicate protein-protein interactions as providing the driving force for cooperativity. First, mutations have been located on the surface of CRP in a putative protein interacting domain that interfere with cooperativity with CytR and with CytR-mediated repression (
      • Sogaard-Andersen L.
      • Mironov A.S.
      • Pedersen H.
      • Sukhodelets V.V.
      • Valentin-Hansen P.
      ). Second, CytR and CRP are reported to mutually antagonize each other's protein binding induced bends in the cytRP sequence (
      • Pedersen H.
      • Sogaard-Andersen L.
      • Holst B.
      • Gerlach P.
      • Bremer E.
      • Valentin-Hansen P.
      ). Since such coupled DNA structural transitions necessarily contribute unfavorably to cooperativity, the driving force for cooperativity in cytRP must be derived from favorable protein-protein contacts. Third, a truncated CytR which is lacking the DNA-binding helix-turn-helix motif was reported to bind to deoP2 in the presence of CRP and further to do so with only moderate reduction in overall affinity (
      • Sogaard-Andersen L.
      • Valentin-Hansen P.
      ) compared with the full-length protein. This would suggest that CytR is primarily a protein-protein bridge, dependent on its interactions with CRP bound to the flanking CRP sites for association with its operator. However, the question remains open how even these favorable protein-protein contacts could possibly compensate for the loss of a direct DNA binding interaction with a Kd in the 10 nM range.
      Previous approaches used to investigate CytR-regulated promoters have not been fully successful in developing an understanding of the macromolecular interactions that regulate transcription. The confusion stems in large part from the fact that only qualitative reasoning has been used to address quantitative questions. For example, most of the in vitro studies have been conducted as combinations of plus/minus the various components (CRP, CytR, cAMP, cytidine, and promoter elements) with insufficient understanding of the mutual effects of interactions between these molecules to know what concentrations are necessary to achieve particular effects. Our immediate goal was to understand how the protein-DNA and protein-protein interactions control the distribution of operator configurations at deoP2. Our approach has been to use DNase footprinting to obtain complete individual site isotherms for binding of CytR and CRP to deoP2, at different configurations of empty and filled sites for the other regulatory protein. From such data, the complete population distribution of promoter configurations can be determined as a function of both CRP and CytR concentration. We anticipated being able to deduce connections between the individual promoter configurations in the distribution and biologically functional states.
      The results indicate a much more complex promoter structure than was previously supposed. Multiple, specific CytR sites are arranged over an extended region of DNA that includes both CRP1 and CRP2 as well as the sequence they flank. CytR binding to these sites mediates both cooperative and competitive interactions with CRP. Together these explain quantitatively the apparent one-way stimulation. Similar specific interactions of CytR with extended DNA sequences have been identified at deoP1 and also near the middle of the approximately 600-bp sequence separating P1 and P2. Comparison of the DNA sequences of these sites further clarifies the CytR binding motif. The distribution of such motifs, both at deoP2 and at other CytR-regulated promoters, define arrays of contiguous and overlapping CytR binding sites. This entirely new phenomenon is consistent with the interpretation that CytR functions as a modulator of CRP-mediated activation. We envision such a function operating at two levels: first as a differential modulator of class I versus class II CRP activation at individual promoters, and second as a differential modulator of activation of the various CytR cistrons.

      DISCUSSION

      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. This illustrates a common strategy by which a small number of transcription factors directs broad gene regulatory programs. Our goal was to provide a quantitative description of these interactions at deoP2, both in order to understand how individual macromolecular interactions and promoter states combine to regulate transcription from deoP2 and to serve as a model for investigation of other CytR-regulated promoters. This has led to the discovery of a feature of deoP2 which has not been described previously for this or other gene regulatory systems and which might lie at the heart of the regulatory mechanism of all CytR-regulated promoters. Specifically, CytR binds to multiple closely spaced or even overlapping sites on DNA with differing affinities and not to just a single operator as previously presumed. Interaction of CytR with these sites provides a complex pattern of both competitive and cooperative interactions between CytR and CRP bound to CRP1 and CRP2.

      Cooperative and Competitive Interactions of CytR

      The initial indication and one functional consequence of these interactions is that while saturation of CRP1 and CRP2 greatly increases the apparent affinity of CytR for its putative operator located between these, saturation by CytR has a much lesser effect on the apparent affinity of CRP for CRP1 and CRP2 (Table I) (
      • Perini L.
      • Doherty E.A.
      • Senear D.F.
      ). A similar effect has been noted also for the nupG promoter (
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ). Because the Gibbs free energy is a thermodynamic function of state, the total free energy change to saturate either of these promoters with both proteins, whether by CRP binding followed by cooperative CytR binding or by the reverse, is necessarily the same. It is evident that the experimental observations can only be rationalized by additional macromolecular interactions, ones that have not been properly accounted.
      Neither contamination of one or another of the protein preparations by another interacting molecule nor some similar experimental artifact provides a plausible explanation; similar observations were made on different regulatory regions by different laboratories using different experimental protocols. On the other hand, CytR binding to additional sites that compete with CRP(cAMP)1 for binding to CRP1 and CRP2 presents a plausible hypothesis based on just a qualitative evaluation of the effects. At high CytR concentration, favorable interactions with CytR would increase the apparent affinity of CRP(cAMP)1 for CRP1 and CRP2. But this would also be partly offset by unfavorable competition between CytR and CRP for binding to overlapping sites at CRP1 and CRP2. By contrast, at high CRP concentration, favorable interactions with CRP bound to CRP1 and CRP2 would increase apparent affinity of CytR for its intervening site, and this would not be offset by unfavorable competitive interactions.
      As our analysis indicates, such competition also accounts quantitatively for the binding. Excellent fits to all of the titration data for both CRP and CytR, whether alone or in combination, and for both wild type and reduced valency deoP2 were obtained by global analysis using simple models for the competing sites. The mathematical formulations considered only thermodynamic properties of the additional CytR sites. These yielded well bounded estimates of CytR affinity for the competing CytR sites even though the data for these sites were not analyzed. These are specific sites for CytR binding. CytR binding to a single operator does not simply nucleate nonspecific binding to adjacent DNA sequences.
      The model's quantitative predictions were tested against three independent criteria and found consistent with the experimental facts in each instance. First, estimated affinities for the hypothesized additional CytR sites match the CytR concentration dependence of the DNase I protection in CRP1 and CRP2. Second, gel mobility shift analysis demonstrated multiple, specific deoP2·CytR complexes, whose populations are consistent with quantitative predictions of the model. Third, under inducing conditions in which binding of cytidine to CytR eliminates positive cooperativity between CRP and CytR (
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ),2 CytR was found to be a competitive inhibitor of CRP binding.

      CytR Binding Sites Upstream from deoP2 Define a Recognition Motif and Demonstrate an Array of Overlapping deoP2 Sites

      In addition to these specific CytR binding sites in the P2 regulatory region, we have identified two other CytR binding loci. These are located about 240 bp upstream from P2 and in the −10 region of deoP1, respectively. These sites are also specific for CytR binding as judged both by the occurrence of localized DNase I protection and by competition with non-regulatory DNA in both footprint and gel shift experiments. In fact, the P2 upstream site has the highest affinity CytR binding that has been reported (cf. Refs.
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ,
      • Holst B.
      • Sogaard A.L.
      • Pedersen H.
      • Valentin-Hansen P.
      ,
      • Pedersen H.
      • Sogaard-Andersen L.
      • Holst B.
      • Gerlach P.
      • Bremer E.
      • Valentin-Hansen P.
      ). Therefore, these DNA sequences constitute specific CytR recognition sequences.
      We have compared these sequences to the regulatory region of deoP2 and other CytR-regulated promoters (Fig. 10) to further refine the consensus CytR recognition motif. Guiding this comparison is a 5-bp motif, TGCAA, previously proposed based on comparative sequence analysis (
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ) and supported by an analysis of mutations that affect CytR binding and regulation of deoP2 (
      • Rasmussen P.B.
      • Sogaard A.L.
      • Valentin-Hansen P.
      ). Pairs of inverted and/or direct repeats of this motif with variable spacing (2-5 bp) are thought to constitute CytR operators in the different CytR-regulated promoters (cf. Refs.
      • Brikun I.
      • Suziedelis K.
      • Stemmann O.
      • Zhong R.
      • Alikhanian L.
      • Linkova E.
      • Mironov A.
      • Berg D.E.
      ,
      • Rasmussen P.B.
      • Sogaard A.L.
      • Valentin-Hansen P.
      and Fig. 10). However, if based on the criterion of most probable nucleotide at each position in these putative CytR operators, then the 6-bp sequence, TTGCAA, defines the consensus. This palindromic motif obviates any distinction between inverted and direct repeats, as previously noted for deoP2 (
      • Rasmussen P.B.
      • Sogaard A.L.
      • Valentin-Hansen P.
      ).
      Figure thumbnail gr10
      Fig. 10CytR-regulated promoter sequences, numbered from +1 at the transcription start site. CytR recognition motifs are in bold with consensus base pairs indicated by larger type size. Putative binding sites are underlined and labeled with a fraction whose numerator is the total number of mismatches in the two 6-bp motifs and whose denominator is the spacing between motifs. Shading indicates the pair of motifs in each promoter previously believed to be the only CytR operator. For reference, the CRP recognition motifs are boxed, and the sites are labeled as CRP1 or CRP2. Below the operators are the sequences of the deo upstream and P1 CytR sites are described in the text. A transition at −23 in tsxP2 (G to A) that affects CytR regulation (see text) is indicated.
      Fig. 10 shows the results when the deoP2 regulatory sequences are searched for both individual matches to the 6-bp motif (with the criterion that at least 4 of 6 bp match) and pairs of such motifs with the requisite spacing (2-5 bp). By either criterion, what emerges is a continuous array of adjacent and overlapping sites, ranging from the promoter distal edge of CRP2 to at least the promoter distal edge of CRP1. The consensus is best matched by the site previously identified as the CytR operator, centered at −70.5 bp. This site coincides with the highest affinity binding we observe by footprint titration. The putative flanking sites in both directions match the consensus less well, consistent with the footprints which indicate decreasing affinity for CytR binding to these sequences.
      The sequences protected from DNase I cleavage by CytR binding to the upstream and P1 sites are also coincident with appropriately spaced pairs of motifs (Fig. 10). These sites both appear to comprise pairs of overlapping CytR binding sites. The P1 site contains a single perfect match to the motif flanked by less perfect matches, only 3 of 6, yet the affinity for CytR binding here is the same as for the −70.5 P2 site. This is significant in relation to the CytR binding observed at CRP1. While there is no pair of motifs in CRP1, individual motifs are arrayed there and even downstream to the −10 promoter element. At least in CRP1, these apparently suffice for weak yet still specific CytR binding, consistent with the protection observed.

      An Array of CytR Binding Sites Is a Common Element of CytR-regulated Promoters

      Arrays of CytR binding sites are not unique to deoP2 but instead are also found in other CytR-regulated promoters (Fig. 10). Evidence of their functional significance in those promoters is abundant. For example, while extended protection patterns in nupG (both DNase I and chemical modification reagents) have been interpreted as resulting from CytR binding to two perfect TGCAA motifs separated by an 11-bp spacer (
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ), this overlooks several additional CytR recognition motifs, including one located in the middle of the proposed 11-bp spacer. Together these define four separate, adjacent, and overlapping binding sites. Because titration studies were not conducted it isn't possible to assess either individual affinities for CytR or the mutual effects of CRP binding and of CytR binding to different sites. However, the array of CytR sites overlaps CRP1, the lower affinity of the two CRP sites in nupG (
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ) and consequently the most easily affected by competition from CytR. A single CytR recognition motif within CRP2 might also constitute a weak binding site, analogous to the situation in deoP2. The structure of nupG is an inversion of deoP2, and similar interactions between CytR and CRP, no doubt, explain the apparent one-way stimulation (
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ).
      Extended binding has been observed also for both cytRP (
      • Pedersen H.
      • Sogaard-Andersen L.
      • Holst B.
      • Gerlach P.
      • Bremer E.
      • Valentin-Hansen P.
      ) and udp (
      • Brikun I.
      • Suziedelis K.
      • Stemmann O.
      • Zhong R.
      • Alikhanian L.
      • Linkova E.
      • Mironov A.
      • Berg D.E.
      ). In both cases, CytR concentrations in the 10 nM range protect a single site. But additional protection is observed at only a few-fold higher CytR concentration, exactly in line with our deoP2 data. The particular protection patterns conform to the distribution of CytR recognition motifs shown in Fig. 10: individual loci mapped by hydroxyl radical footprinting in the −50 to −70 region of cytRP (
      • Pedersen H.
      • Sogaard-Andersen L.
      • Holst B.
      • Gerlach P.
      • Bremer E.
      • Valentin-Hansen P.
      ) and DNase I protection of the entire CRP1/CRP2 region of udp (
      • Brikun I.
      • Suziedelis K.
      • Stemmann O.
      • Zhong R.
      • Alikhanian L.
      • Linkova E.
      • Mironov A.
      • Berg D.E.
      ). This additional protection has been interpreted as aggregation of CytR on the DNA (
      • Pedersen H.
      • Sogaard-Andersen L.
      • Holst B.
      • Gerlach P.
      • Bremer E.
      • Valentin-Hansen P.
      ). However, since CytR does not aggregate in solution at sub-μM concentration3 this should be interpreted as protein binding to DNA rather than protein aggregation per se. It is also noteworthy that gel mobility shift experiments on these systems have not provided evidence for multiple liganded complexes, whether in the presence or absence of CRP(cAMP)1 binding. However, none of these experiments (cf. Refs.
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ,
      • Brikun I.
      • Suziedelis K.
      • Stemmann O.
      • Zhong R.
      • Alikhanian L.
      • Linkova E.
      • Mironov A.
      • Berg D.E.
      ,
      • Pedersen H.
      • Sogaard-Andersen L.
      • Holst B.
      • Gerlach P.
      • Bremer E.
      • Valentin-Hansen P.
      ,
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ) was conducted at saturating CytR concentration.
      TsxP2 also offers evidence of similar interactions with CytR. CytR is reported to protect only a 20-bp sequence (
      • Gerlach P.
      • Soogard-Andersen L.
      • Pedersen H.
      • Martinussen J.
      • Valentin-Hansen P.
      ) corresponding to the putative CytR operator that overlaps CRP1 (Fig. 10). Because these experiments used extracts of unknown CytR concentration, they don't address whether additional lower affinity sites also exist. But when CRP is added to this same CytR concentration, cooperative binding of CytR and CRP produces a footprint that extends beyond CRP1 to the −10 region of the promoter. The region protected is much larger than by either protein alone, and the protection pattern in CRP1 is distinctly different from that produced by CRP binding alone. Supporting the interpretation that this represents additional CytR binding is a promoter mutation that greatly reduces CytR-mediated repression (
      • Gerlach P.
      • Soogard-Andersen L.
      • Pedersen H.
      • Martinussen J.
      • Valentin-Hansen P.
      ). The mutation, located in the protected region downstream from CRP1 (Fig. 10), is in a CytR recognition motif.

      Energetics of Competition and Cooperativity

      The model we developed to analyze our binding data was developed as the simplest model to rationalize the observed energetics. It clearly oversimplifies the actual situation. As there appear to be more than three CytR binding sites, the saturating CytR stoichiometry, whether in the presence or absence of CRP(cAMP)1, isn't obvious. It shouldn't be a surprise that the gel mobility shift assay indicates more than three ligation states (Fig. 8). Also since adjacent sites overlap, CytR binding to these should be self-competitive, a feature we did not incorporate. Perhaps this accounts for the small difference between predicted and observed competitive binding curves in Fig. 9. In recognition of these facts, our formulation of competition should be considered as a phenomenological description only.
      On the other hand, the free energy changes for cooperativity between CytR and CRP are independent of the particular model and so appear to provide accurate descriptions of these interactions. It seems significant that ΔG13 and ΔG23 are equal. If direct protein-protein interactions provide the driving force for cooperativity, as has been presumed (23 58), then CytR bound to the intervening site must make substantially identical interactions with CRP, whether bound to CRP1 or to CRP2. This situation is reminiscent of the bacteriophage λ cI repressor which similarly makes identical pairwise cooperative interactions between adjacent sites with spacing ranging from 3 to 7 bps (
      • Johnson A.D.
      • Poteete A.R.
      • Lauer G.
      • Sauer R.T.
      • Ackers G.K.
      • Ptashne M.
      ). Second, it may also be significant that the three-way cooperativity, i.e. with both CRP sites filled and CytR bound in between, equals the additive sum of free energy changes for these pairwise interactions. This suggests that CytR bound to the intervening site simultaneously contacts CRP bound to both CRP1 and CRP2 and with no influence of either pairwise interaction on the other. We might refer to these as “complementary pairwise” interactions by comparison to the cI repressor pairwise interactions that are not independent of one another (
      • Senear D.F.
      • Ackers G.K.
      ,
      • Johnson A.D.
      • Poteete A.R.
      • Lauer G.
      • Sauer R.T.
      • Ackers G.K.
      • Ptashne M.
      ) and are referred to as “alternate pairwise.”
      How can CytR can make the same interaction with CRP bound to CRP1 and CRP2 given spacers of 15 and 8 bps? There are several possibilities. First, CytR might have substantial structural flexibility as might result if its DNA binding head and inducer binding domain are only loosely tethered to one another. Such flexibility could also be invoked to explain CytR's ability to bind pairs recognition motifs with variable spacing of 2-5 bp in different operators. The structures of two homologues, PurR and LacR, are known from crystallographic studies (
      • Friedman A.M.
      • Fischmann T.O.
      • Steitz T.A.
      ,
      • Lewis M.
      • Chang G.
      • Horton N.C.
      • Kercher M.A.
      • Pace H.C.
      • Schumacher M.A.
      • Brennan R.G.
      • Lu P.
      ,
      • Schumacher M.A.
      • Choi K.Y.
      • Zalkin H.
      • Brennan R.G.
      ,
      • Schumacher M.A.
      • Choi K.Y.
      • Lu F.
      • Zalkin H.
      • Brennan R.G.
      ). In these cases, the DNA binding helix-turn-helix and core domains are connected by hinge helices which form stable structures only when the repressors are bound to DNA (
      • Lewis M.
      • Chang G.
      • Horton N.C.
      • Kercher M.A.
      • Pace H.C.
      • Schumacher M.A.
      • Brennan R.G.
      • Lu P.
      ,
      • Schumacher M.A.
      • Choi K.Y.
      • Lu F.
      • Zalkin H.
      • Brennan R.G.
      ). Perhaps CytR differs from those by not forming stable structures even when bound. Second, there might be a specific three protein, CRP·CytR·CRP complex that is asymmetric with respect to its interactions with DNA. Third, interaction with CRP might involve a repositioning of CytR on the DNA as has been suggested independently (
      • Mollegaard N.E.
      • Rasmussen P.B.
      • Valentin H.P.
      • Nielsen P.E.
      ). Our data do not address this question; further investigation is necessary.

      Role of Competition and Cooperativity in deoP2 Regulation

      The important question remaining is how these multiple competitive and cooperative interactions between CytR and CRP regulate transcription. The energetics alone cannot provide a definitive answer but do suggest possibilities (and eliminate others). The common perception is that heterologous cooperativity serves to recruit CytR as CRP fills the CRP sites (
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ,
      • Pedersen H.
      • Dall J.
      • Dandanell G.
      • Valentin-Hansen P.
      ). In this view, CytR is presumed to be an ineffective inhibitor of transcription in the absence of CRP binding only because its free concentration is insufficient to fill the site. The total effect of cooperativity with both CRP sites on CytR affinity is 100-1000-fold in different promoters and conditions (cf. Table I and Refs.
      • Pedersen H.
      • Sogaard-Anderson L.
      • Holst B.
      • Valentin-Hansen P.
      ,
      • Holst B.
      • Sogaard A.L.
      • Pedersen H.
      • Valentin-Hansen P.
      ) or just about sufficient to provide such a switch. But simple recruitment of this sort makes little functional sense, as it would yield only two significantly populated states, i.e. CRP and CytR sites empty or CRP and CytR sites filled. The result would be two-state, on or off, regulation. Since the combination of appropriate promoter (strong or weak) and either protein alone is sufficient to provide such regulation, it isn't clear why a more complex mechanism would have evolved.
      On the other hand, considering both cooperativity and competition together suggests a substantially more complex role for CytR. We start with the premise that CytR is a modulator of CRP activation rather than a repressor per se, a view consistent with the fact that CytR is ineffective in the absence of CRP binding. In this view the state of CRP activation is as important as occupancy by CytR. The tandem CRP sites and pairwise heterologous cooperativity are crucial to such a role as is the suggestion that the molecular mechanism of CRP activation differs at class I versus class II sites (
      • Zhou Y.
      • Pendergrast P.S.
      • Bell A.
      • Williams R.
      • Busby S.
      • Ebright R.H.
      ). CRP is proposed to activate transcription by making direct protein-protein contacts with the C-terminal domain of the α-subunit of RNAP, thereby directing interactions between RNAP and otherwise nonspecific DNA sequences (
      • Busby S.
      • Ebright R.H.
      ), substituting for the UP promoter element found in some particularly active genes (cf. Ref.
      • Ross W.
      • Gosink K.K.
      • Salomon J.
      • Igarashi K.
      • Zou C.
      • Ishihama A.
      • Severinov K.
      • Gourse R.L.
      and references therein). A surface loop of CRP that is involved in the protein-protein contact has been identified (66). The subunit of CRP that contacts the α-RNAP subunit depends on the promoter architecture; it is the proximal subunit at class I sites (CRP2) but the distal subunit at class II sites (
      • Zhou Y.
      • Pendergrast P.S.
      • Bell A.
      • Williams R.
      • Busby S.
      • Ebright R.H.
      ). In either case, the new RNAP-DNA interaction would be in the sequence between CRP2 and CRP1, precisely where CytR binds. In fact, RNAP has been reported to make minor groove contacts in this region, dependent on CRP binding (
      • Mollegaard N.E.
      • Rasmussen P.B.
      • Valentin H.P.
      • Nielsen P.E.
      ). CytR should function as an RNAP antagonist by competing with this upstream RNAP-DNA interaction.
      At low CytR concentration, increasing cAMP concentration directs a two-stage activation: first, of class I as CRP2 fills and second, of class II as CRP1 subsequently fills. Because heterologous cooperative interactions are complementary pairwise in nature, recruitment of CytR via heterologous cooperativity is also a two-stage process. First, pairwise cooperativity between CytR and CRP(cAMP)1 bound to CRP2 yields about a 10-fold increase in effective CytR affinity. Although a significant effect, this alone is not sufficient to shut down CRP2-mediated activation. However as CRP(cAMP)1 subsequently binds CRP1, the additional pairwise cooperative interaction should now cause CytR to compete effectively for CRP2-RNAP interactions and so tend to shut down CRP2-mediated activation.
      We suggest that cooperative CytR binding has very different effects on class I CRP activation mediated by CRP2 versus class II activation mediated by CRP1. Under these conditions of low CytR concentration, CytR might be an ineffective repressor of the latter. As such, CytR would act initially as a switch from one kinetic mode of CRP activation to another. There are two reasons for thinking this should be so. First, activation at class II sites has been reported which appears to bypass these critical interactions between α-RNAP and its interactions with DNA (
      • Zhou Y.
      • Pendergrast P.S.
      • Bell A.
      • Williams R.
      • Busby S.
      • Ebright R.H.
      , 67) but which instead involves interactions with other components of RNAP. CytR binding should not compete with such a mechanism. Second, while mutant deoP2 with only CRP1 functional (i.e. CRP2) displays nearly the same extent of CRP activation (presumably class II-mediated) as wild type, it is not effectively repressed by CytR (
      • Sogaard-Andersen L.
      • Mollegaard N.E.
      • Douthwaite S.R.
      • Valentin-Hansen P.
      ). Occupancy of the DNA sites by CytR site cannot account for this. At the high CRP(cAMP)1 concentration required to significantly load CRP1, the net effect of cooperativity and competition produces only a slight difference in CytR occupancy whether deoP2 is wild type or CRP2 (Table I).
      Additional CytR sites and competition with CRP might play several roles in such a scheme. The primary role, regardless of specific mechanism, is probably to provide exactly the observed one-way stimulation. This is accomplished by partially decoupling CRP binding from CytR binding. Simple cooperativity is of course a two-way street, and as such, CytR would be a net recruiter of CRP as well as the reverse. This would proportionally diminish the effectiveness of CytR as a negative regulator. On the other hand, the combined effect of competition and cooperativity on CRP binding to both CRP1 and CRP2 is small (Fig. 7). Consequently, CytR is not a significant net recruiter of CRP. Thus, while repression, which we view as modulation of activation, depends on a complex interplay between cAMP, CytR, and cytidine concentrations, the initial activation is controlled only by cAMP concentration and is largely independent of CytR and cytidine. The second, simpler role of competition comes into play at very high CytR concentrations where CytR simply becomes a net inhibitor of CRP binding. This effect occurs first at CRP2 due to the higher affinity of CytR for its competing site(s), thus helping to facilitate the switch from class I to class II CRP activation. However, it also provides a final, cutoff of CRP-mediated activation at sufficient CytR concentration.
      Finally, one can speculate on the role for overlapping multiple CytR sites (Fig. 10). Presumably these cannot be occupied simultaneously. Repositioning of CytR between these sites would be possible, particularly in configurations with only one CRP site (CRP1 or CRP2) occupied. Repositioning might provide a configuration in which RNAP makes favorable contacts with upstream DNA sequences even while CytR is still bound. These RNAP-DNA interactions would now compete only with the difference in free energy changes (intrinsic and cooperative) to bind CytR at one location versus another. The free energy penalty is substantially less than if CytR must be displaced entirely in order for RNAP to interact with upstream DNA sequences. Thus, repositioning of CytR could have a profound effect on RNAP-CRP-DNA interactions and therefore on the effectiveness of CytR-mediated repression.

      Role of Competition and Cooperativity in Differential Regulation

      Bacterial promoters are often described as having a small number of functional states, e.g. basal, activating, repressing, and inducing. The accuracy of this view is questionable, even when considering simple catabolic operons which need only be responsive to availability and requirement for an individual metabolite. Coordinate regulation of unlinked operons poses an even more complex question. CytR regulates expression from different cistrons of enzymes and transport proteins involved in both catabolism and recycling of nucleosides. To balance the flux of nucleoside metabolites, regulation of both absolute and relative enzyme levels must be responsive to widely varying metabolic conditions. Regardless of the particular details, the defining feature of deoP2 regulation is a complex and highly interdependent array of ligation states. This in turn controls the distribution of a large number of functional states. The reason for this would appear to be to provide continuous modulation of expression of each of the genes as a multi-variant function of the metabolic state of the cell.
      Based both on the structures of the promoters and on experimental observations made on other promoters, it seems evident that cooperative and competitive interactions such as we detail here are not unique to deoP2. Instead this appears to be a general feature of CytR-mediated control of gene expression. In comparing the structures of the different CytR-regulated promoters, most have similar tandem CRP sites. What appears to distinguish them are their very different arrays of CytR binding sites. These presumably result in very different patterns of cooperative and competitive interactions. While one necessarily looks to unique features of the different promoters to provide for their differential regulation, the existence of a common theme or organizing principle to those differences is inherently attractive. Therefore, while we cannot predict the specific effects of these different arrays of CytR sites on regulation of other promoters, we can reasonably speculate that the different balance of similar effects is critically involved in the mechanism of their differential regulation.

      Acknowledgments

      We acknowledge our collaborator Steven A. Short both for his invaluable insight and for supplying us with the CytR expression strain and deo containing plasmid. We thank Jim Lee and Angela Gronenborn for the CRP expression strain and Thomas Heyduk for advice on purifying CRP. We thank Henrik Pedersen and Poul Valentin-Hansen for sharing their nupG manuscript prior to its publication.

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