An Unusual Pattern of CytR and CRP Binding Energetics at Escherichia coli cddP Suggests a Unique Blend of Class I and Class II Mediated Activation

Abstract

Two transcription factors, CRP and CytR, mediate positive and negative control of nine cistrons involved in nucleoside catabolism and recycling in Escherichia coli. The ability of multiple transcription factors to combine in different ways to confer differential gene regulation is of significant interest in both prokaryotic and eukaryotic gene regulation. Analysis of cooperative interactions between CytR and CRP at the deoP2 and udpP promoters has implicated the importance of promoter architecture in controlling repression and induction. These studies have also identified competition between CytR and CRP as an additional contributor to differential regulation. The pattern and energetics of CytR and CRP interactions at the cdd promoter, the most strongly activated of the CytR-regulated promoters, have been delineated using DNase I footprinting. Surprisingly, CRP has greater affinity for the promoter proximal site at cddP, CRP1, than for the distal site, CRP2, in contrast to promoters studied previously. This difference is a major contributor to unusually high CRP-mediated activation of cddP. Additionally, while cytidine binding to CytR nearly eliminates the pairwise interactions between CytR and CRP bound at CRP1, it has little effect on pairwise cooperativity between CytR and CRP bound at CRP2 or as a consequence on the overall cooperativity of the three-protein complex in which CRP is bound to both sites. The effect of cytidine binding on cooperativity differs between the three promoters studied thus far. We propose that the different patterns of interaction reflect the spacing between CytR half-sites and the location of the CytR operator in relation to the two CRP sites.

Comparative Genomics of CytR, an Unusual Member of the LacI Family of Transcription Factors

“…The most recent description implies only octameric repeats with a spacer allowing both of them to be situated on the same side of the DNA-helix, with the spacer being less than 4–5 nucleotides or roughly a helical turn, that is 10–11 nucleotides [18], [20]. The current experimental data agree with this description [13], [29], [39]. The distances of about two or three helical turns were experimentally proven to be possible [18] but so far have not been observed in nature.…”

“…The number of CRP-binding sites (O CRP ) per CytR-binding site (O CYTR ) varies from one to three [17]: one, as in the cytR promoter [6]; two, as in the majority of cases; or three, as in the cdd promoter [8], [17], [29]. This might indicate different structures of the CRP-CytR complex or repositioning of the CRP dimers upon interaction.…”

“…This might indicate different structures of the CRP-CytR complex or repositioning of the CRP dimers upon interaction. In most promoters, CRP has a stronger affinity to the distal operator O CRP D [30], with an exception being cddP , where CRP binds stronger to its proximal operator O CRP P [29]. An important requirement is that at least one CRP-operator has to be situated at a distance not exceeding 5 nucleotides to the corresponding CytR-operator [20], with the position of the O CYTR operator being not symmetric relative to the flanking O CRP operators [17].…”

Different Requirements for σ Region 4 in BvgA Activation of the Bordetella pertussis Promoters Pfim3 and PfhaB

“…The positions of the BvgA~P binding sites within P fhaB are suggestive of a combination of Class I and Class II activation 16; 17 . In such an arrangement, σ Region 4 is typically required for activation, providing a direct contact with the promoter proximal activator (reviewed in 14 ).…”

“…Most of the virulence genes in B. pertussis have an organization that resembles that seen at ClassI/II activated promoters 16; 17 . For example, the fhaB gene is activated by three dimers of BvgA~P, which bind from −95 to −38 relative to the transcription start site 18 (Fig.…”