Poul Valentin-Hansen

A flexible partnership: the CytR anti-activator and the cAMP–CRP activator protein, comrades in transcription control

A vital point in gene regulation is control at the level of transcription initiation. Recent research has established that this regulation can involve sophisticated networks of interacting proteins that modulate the activity of the transcription machinery by DNA looping, direct protein–protein interactions or changing DNA topology in the promoter region. This MicroReview focuses on our investigations of a relatively simple prokaryotic gene regulatory system, the Escherichia coli CytR regulon, which exhibits a number of these features. This work has opened the door to the molecular understanding of how a prokaryotic repressor can be correctly positioned at specific DNA sequences with the help of a global activator, and how the repressor subsequently inhibits factor‐dependent transcription initiation.

Protein‐induced fit: the CRP activator protein changes sequence‐specific DNA recognition by the CytR repressor, a highly flexible LacI member

The CytR repressor and the cAMP receptor protein (CRP) bind cooperatively to several promoters in Escherichia coli to repress transcription initiation. The synergistic binding is mediated by protein–protein interactions between the two regulators. Here, in vitro selection experiments have been used to examine the DNA‐binding characteristics of CytR, by itself and when co‐binding with cAMP–CRP. We show that the optimal CytR‐binding site consists of two octamer repeats, in direct or inverted orientation, and separated by 2 bp. However, when co‐binding with cAMP–CRP, CytR instead recognizes inverted repeats separated by 10–13 bp, or direct repeats separated by 1 bp. The configurations of the latter set of operators correlate well with the configurations of natural CytR targets. Thus, cAMP–CRP induces conformational changes in CytR so that the repressor fits the natural targets. Most strikingly, CytR can adopt widely different conformations that are equally favored energetically for complex formation with cAMP–CRP. We propose that this structural adaptability is essential for CytR repression of promoters with diverse architectures. We discuss these novel concepts in the context of the CRP/CytR regulatory system, as well as the structural and functional implications for multiprotein–DNA complex formation in general.

Regulation of the deo operon in Escherichia coli

SummaryThe synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes.A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate.Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants.Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.

Protein-protein interactions in gene regulation: The cAMP-CRP complex sets the specificity of a second DNA-binding protein, the CytR repressor

Maximal repression by the CytR protein depends on the formation of nucleoprotein complexes in which CytR interacts with DNA and with cAMP-cAMP receptor protein (CRP). Here we demonstrate that CytR regulates transcription from deoP2 promoters in which the entire CytR recognition sequence has been eliminated. Furthermore, CytR proteins deleted for the DNA-binding domain repress deoP2 in vivo and interact with deoP2 in vitro in a strictly cAMP-CRP-dependent fashion. These experiments show that the site of action of CytR can be specified by protein-protein interactions to cAMP-CRP, whereas CytR-DNA interactions may primarily serve to stabilize the nucleo-protein complex. This type of specificity mechanism may represent a general concept in the recruitment of DNA-binding proteins in combinatorial regulatory systems.

DNA-binding characteristics of the Escherichia coli CytR regulator: a relaxed spacing requirement between operator half-sites is provided by a flexible, unstructured interdomain linker.

The Escherichia coli CytR regulator belongs to the LacI family of sequence‐specific DNA‐binding proteins and prevents CRP‐mediated transcription in the CytR regulon. Unlike the other members of this protein family, CytR binds with only modest affinity to its operators and transcription repression thus relies on the formation of nucleoprotein complexes with the cAMP–CRP complex. Moreover, CytR exhibits a rotational and translational flexibility in operator binding that is unprecedented in the LacI family. In this report we examined the effect of changing the spacing between CytR half‐operators on CytR regulation in vivo and on CytR binding in vitro. Maximum repression was seen with the short spacing variants: repression peaks when the half‐operators lie on the same face of the DNA helix. Repression was retained for most spacing variants with centre separations of half‐operators ≤u20033 helical turns. Our data confirm and extend the view that CytR is a highly flexible DNA binder that can adapt many different conformations for co‐operative binding with CRP. Furthermore, limited proteolysis of radiolabelled CytR protein showed that the interdomain linker connecting the DNA binding domains and the core part of CytR does not become structured upon DNA binding. We conclude that CytR does not use hinge α‐helices for minor groove recognition. Rather, CytR possesses a highly flexible interdomain linker that allows it to form complexes with CRP at promoters with quite different architecture.

Transcription of rpoH, encoding the Escherichia coli heat-shock regulator σ32, is negatively controlled by the cAMP-CRP/CytR nucleoprotein complex

In Escherichia coli, the rpoH gene encoding the essential heat‐shock regulator σ32, is expressed in a complex manner. Transcription occurs from four promoters (P1, P3, P4 and P5) and is modulated by several factors including (i) two σ factors (σ70 and σE); (ii) the global regulator CRP; and (iii) the DnaA protein. Here, a further dissection of the rpoH regulatory region has revealed that an additional transcription control exists that appears to link rpoH expression to nucleoside metabolism. The cAMP–CRP complex and the CytR anti‐activator bind co‐operatively to the promoter region forming a repression complex that overlaps the σE‐dependent P3 promoter and the σ70‐dependent P4 and P5 promoters. During steady‐state growth conditions with glycerol as the carbon and energy source, transcription from P3, P4 and P5 is reduced ≈threefold by CytR, whereas transcription from the upstream promoter, P1, appears to be unaffected. Furthermore, in strains that slightly overproduce CytR, transcription from P3, P4 and P5 is reduced even further (≈10‐fold), and repression can be fully neutralized by the addition of the inducer cytidine to the growth medium. In the induced state, P4 is the strongest promoter and, together with P3 and P5, it is responsible for most rpoH transcription (65–70%). At present, CytR has been shown to ‘fine tune’ transcription of two genes (rpoH and ppiA) that are connected with protein‐folding activities. These findings suggest that additional assistance in protein folding is required under conditions in which CytR is induced (i.e. in the presence of nucleosides).

Protein-protein communication: structural model of the repression complex formed by CytR and the global regulator CRP.

The cAMP receptor protein (CRP) and the LacI-related CytR antiactivator bind cooperatively to adjacent DNA sites at or near promoters, an interaction that involves direct protein contacts. Here, we identify a collection of amino acid substitutions in CytR that reestablish protein-protein communication to mutant CRP proteins specifically defective in cooperative binding with wild-type CytR. To assess the location and spatial arrangement of these substitutions, we built a three-dimensional model of CytR based on the recent X-ray structure of the highly homologous PurR repressor bound to DNA. This approach enables us to specify the patch on CytRs surface that contacts CRP. Furthermore, our results permit the construction of a three-dimensional structure of the higher order nucleoprotein complex formed by CytR and CRP.

Analysis of the tsx gene, which encodes a nucleoside-specific channel-forming protein (Tsx) in the outer membrane of Escherichia coli

The tsx gene of Escherichia coli encodes an outer membrane protein, Tsx, which constitutes the receptor for colicin K and bacteriophage T6, and functions as a substrate-specific channel for nucleosides and deoxynucleosides. The mini-Mu element pEG5005 was used to prepare a gene bank in vivo, and this bank was used to identify T6-sensitive strains carrying the cloned tsx gene. Subcloning of the tsx gene into the multicopy plasmid, pBR322, resulted in a strong overproduction of Tsx. The sequence of a 1477-bp DNA segment containing tsx and its flanking regions was determined. An open reading frame (ORF) was found which was followed by a pair of repetitive extragenic palindromic sequences. This ORF translated into a protein of 294 amino acids (aa), the first 22 aa of which showed the characteristic features of a bacterial signal sequence peptide. The putative mature form of Tsx is composed of 272 aa with a calculated Mr of 31418. The aa sequence of Tsx shows an even distribution of charged residues (52 aa) and lacks extensive hydrophobic stretches. No significant homologies of Tsx to the channel-forming proteins OmpC, OmpF, PhoE and LamB from the E. coli outer membrane were detected. Using nuclease S1, we identified two transcription start points for the tsx mRNA which were separated by approx. 150 bp. Genetic data suggest that the synthesis of the larger mRNA species is directed by a weak promoter (P1) that is controlled by the DeoR repressor, whereas the smaller mRNA species is directed by the main promoter P2, which is negatively controlled by the CytR repressor and positively affected by the cyclic AMP/catabolite activator protein complex.

The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter.

Transcription initiation at CytR regulated promoters in Escherichia coli is controlled by a combinatorial regulatory system in which the cAMP receptor protein (CRP) functions as both an activator and a co‐repressor. By combining genetic studies and footprinting analyses, we demonstrate that regulated expression of the CytR controlled cdd promoter requires three CRP‐binding sites: a high affinity site (CRP‐1) and two overlapping low affinity sites (CRP‐2 and CRP‐3) centred at positions −41, −91 and −93, respectively. In the absence of CytR, cAMP‐CRP interacts at one set of sites (CRP‐1 and CRP‐2) and both of these binding sites are required for full promoter activation. In the presence of CytR, however, the two regulators bind cooperatively to cddP forming a nucleoprotein complex in which cAMP‐CRP binds to CRP‐1 and CRP‐3 and CytR occupies the sequence between these sites. Thus, association of the two regulators involves a repositioning of the cAMP‐CRP complex. Moreover, mutant cdd promoters in which CRP‐2 and CRP‐3 have been deleted are partially regulated by CytR, and cAMP‐CRP and CytR still bind cooperatively to these promoters. These findings provide clues to an understanding of how cAMP‐CRP and CytR interact at a structurally diverse set of promoters.

A novel function of the cAMP-CRP complex in Escherichia coli : cAMP―CRP functions as an adaptor for the CytR repressor in the deo operon

Unlike classical bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression. Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP‐CRP and CytR bind co‐operatively to deoP2. Using mutant promoters we show that tandem, properly spaced DNA‐bound cAMP‐CRP complexes are required for this co‐operative binding. These data suggest that CytR forms a bridge between tandem cAMP‐CRP complexes, and that cAMP‐CRP ftjnctions as an adaptor for CytR. The implications of this new version of negative control in E. coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed.