Catabolite Activator Protein Research Articles

Lac and λ repressors relieve silencing of the Escherichia coli bgl promoter. activation by alteration of a repressing nucleoprotein complex

The Escherichia coli bgl promoter is kept in a repressed state by silencer sequences which flank the promoter and by the histone-like protein H-NS. Silencing of the bgl promoter is likely due to the formation of a repressing nucleoprotein complex of which H-NS is an essential component. Here, we show that silencing is abolished by the binding of Lac or λ repressors to their respective operators that were inserted within the bgl upstream silencer. Efficient activation of bgl operon transcription by Lac and λ repressors was independent of the position and phasing of the operators with respect to the promoter. Activation by Lac and λ repressors as shown here is unprecedented. We conclude that the activation of bgl transcription by both repressors is achieved by a novel mechanism, that is by alteration of the repressing nucleoprotein complex rather than by protein-protein interactions with RNA polymerase and the catabolite activator protein, CAP.

Coupling physiology and gene regulation in bacteria: the phosphotransferase sugar uptake system delivers the signals.

In many bacteria a crucial link between metabolism and regulation of catabolic genes is based on the phosphotransferase sugar uptake system (PTS). We summarize the mechanisms of the signaling pathways originating from PTS and leading to regulation of transcription. A protein domain, called PTS regulation domain (PRD), is linked to many antiterminators and transcriptional activators and regulates their activity depending on its state of phosphorylation. Two sites can be phosphorylated in most PRDs: HPr-dependent modification at one site leads to activation while enzyme II dependent phosphorylation of the other site renders it inactive. In addition, PTS components are used to generate cofactors for regulators of transcription. The paradigm is the enzyme II dependent activity of adenylate cyclase determining the cyclic AMP level in Escherichia coli and thereby the activity of the catabolite activator protein. In many gram-positive bacteria catabolite repression is mediated by the catabolite control protein CcpA, which requires HPr Ser-46 phosphate as a cofactor to regulate transcription of catabolic genes. HPr Ser-46 phosphate is produced by HPr kinase, the activity of which is under metabolic control via the concentrations of glycolytic intermediates. These recent results establish a multifaceted regulatory role for PTS in addition to its well-established function in active sugar uptake.

CAP, the −45 region, and RNA polymerase: three partners in transcription initiation at lacP1 in Escherichia coli

The lac operon of Escherichia coli is positively regulated by the catabolite activator protein (CAP) bound upstream of the −45 region (CAP binding is centered at −61.5; the −45 region extends from −50 to −38). Certain mutations within the −45 region generate sequences that resemble UP elements in base composition and mimic the stimulation by the rrnBP1 UP element, yielding up to 15-fold stimulation in vivo. These −45 region “UP mutants” are compromised in their CAP stimulation. CAP and UP elements do not act in a fully additive manner in vivo at the lac operon. Transcription assays with the wild-type lac promoter and an UP mutant of lac indicate that CAP and UP DNA also fail to act in a completely additive manner in vitro. RNA polymerase can stabilize CAP binding to promoter DNA with a −45 region UP element against a heparin challenge. This shows that CAP and the UP DNA do not compete for the α-CTD as a mechanism for their lack of additivity. CAP and UP elements both demonstrate decreased stimulation of transcription as RNA polymerase concentration is increased from 0.05 to 10 nM in in vitro transcription experiments. In addition CAP also stimulates transcription in a manner that does not decrease as RNA polymerase is varied over this concentration range. This invariable stimulation is by two- to threefold and occurs both in vivo and in vitro. It is not dependent upon the α-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159. This two- to threefold invariable CAP stimulation appears to depend on the −45 region sequence as our −45 region mutants demonstrate different responses to HL159 CAP stimulation in vivo.

Measurement of the DNA bend angle induced by the catabolite activator protein using Monte Carlo simulation of cyclization kinetics

A Monte Carlo simulation method for studying DNA cyclization (or ring-closure) has been extended to the case of protein-induced bending, and its application to experimental data has been demonstrated. Estimates for the geometric parameters describing the DNA bend induced by the catabolite activator protein (CAP or CRP) were obtained which correctly predict experimental DNA cyclization probabilities ( J factors), determined for a set of 11 150 to 166 bp DNA restriction fragments bearing A tracts phased against CAP binding sites. We find that simulation of out-of-phase molecules is difficult and time consuming, requiring the geometric parameters to be optimized individually rather than globally. A wedge angle model for DNA bending was found to make reasonable predictions for the free DNA. The bend angle in the CAP-DNA complex is estimated to be 85 to 90°, in agreement with estimates from gel electrophoresis and X-ray co-crystal structures. Since the DNA is found to have a pre-existing bend of 15°, the change in bend angle induced by CAP is 70 to 75°, in agreement with an estimate from topological measurements. We find evidence for slight (∼10°) unwinding by CAP. The persistence length and helical repeat of the unbound portion of the DNA are in accord with literature-cited values, but the best-fit DNA torsional modulus C is found to be 1.7(±0.2) × 10 −19 erg · cm, versus literature estimates and best-fit values for the free DNA of 2.0 × 10 −19 to 3.4 × 10 −19 erg · cm. Simulations using this low value of C predict that cyclization of molecules with out-of-phase bends proceeds via undertwisting or overtwisting of the DNA between the bends, so as to align the bends, rather than through conformations with substantial writhe. We present experiments on the topoisomers formed by cyclization with CAP which support this conclusion, and thereby rationalize the surprising result that cyclization can actually be enhanced by out-of-phase bends if the twist required to align the bends improves the torsional alignment of the ends. The relationship between the present work and previous studies on DNA bending by CAP is discussed, and recommendations are given for the efficient application of the cyclization/simulation approach to DNA bending.

A State-independent Interaction between Ligand and a Conserved Arginine Residue in Cyclic Nucleotide-gated Channels Reveals a Functional Polarity of the Cyclic Nucleotide Binding Site

Activation of cyclic nucleotide-gated channels is thought to involve two distinct steps: a recognition event in which a ligand binds to the channel and a conformational change that both opens the channel and increases the affinity of the channel for an agonist. Sequence similarity with the cyclic nucleotide-binding sites of cAMP- and cGMP-dependent protein kinases and the bacterial catabolite activating protein (CAP) suggests that the channel ligand binding site consists of a beta-roll and three alpha-helices. Recent evidence has demonstrated that the third (or C) alpha-helix moves relative to the agonist upon channel activation, forming additional favorable contacts with the purine ring. Here we ask if channel activation also involves structural changes in the beta-roll by investigating the contribution of a conserved arginine residue that, in CAP and the kinases, forms an important ionic interaction with the cyclized phosphate of the bound ligand. Mutations that conserve, neutralize, or reverse the charge on this arginine decreased the apparent affinity for ligand over four orders of magnitude but had little effect on the ability of bound ligand to open the channel. These data indicate that the cyclized phosphate of the nucleotide approaches to within 2-4 A of the arginine, forming a favorable ionic bond that is largely unaltered upon activation. Thus, the binding site appears to be polarized into two distinct structural and functional domains: the beta-roll stabilizes the ligand in a state-independent manner, whereas the C-helix selectively stabilizes the ligand in the open state of the channel. It is likely that these distinct contributions of the nucleotide/C-helix and nucleotide/beta-roll interactions may also be a general feature of the mechanism of activation of other cyclic nucleotide-binding proteins.

Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein.

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.

The question of long-range allosteric transitions in DNA.

The question of long-range allosteric transitions of DNA secondary structure and their possible involvement in transcriptional activation is discussed in the light of new results. A variety of recent evidence strongly supports a fluctuating long-range description of DNA secondary structure. Balanced equilibria between two or more different secondary structures, and the occurrence of very large domain sizes, have been documented in several instances. Long-range allosteric effects stemming from changes in sequence or secondary structure over a small region of the DNA have been observed to extend over distances up to hundreds of base pairs in some cases. The discovery that coherent bending strain beyond a threshold level in small (N < or = 250 base pairs (bp)] circular DNAs significantly alters the DNA secondary structure has important implications, especially for transcriptional activators that either bend the DNA directly or are involved in the formation of DNA loops of sufficiently small size (N < or = 250 bp). Whether the RNA polymerase is activated primarily via protein: protein contacts, as is widely believed, or instead via a bend-induced allosteric transition of the DNA in such a small loop, is now an open question. Binding of the transcriptional activator Sp1 to linear DNA induces a remarkably long-range change in its secondary structure, and catabolite activator protein binding to a supercoiled DNA behaves similarly, though possibly for different reasons. Compelling evidence for a bend-induced long-range structural transmission effect of the transcriptional activator integration host factor on RNA polymerase activity was recently reported. These results may augur a new paradigm in which allosteric transitions of duplex DNA, as well as of the proteins, are involved in the regulation of transcription.

Transcription Activation at Class II CAP-Dependent Promoters: Two Interactions between CAP and RNA Polymerase

At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase. We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase α subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase α subunit N-terminal domain. We further show that the two interactions affect different steps in transcription initiation. Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence.

Photoaffinity labelling of a DNA-binding site on the globular domain of histone H5.

We have labelled a DNA-binding site on the globular domain of histone H5 (GH5) by ultraviolet-activated cross-linking of a self-complementary 5-bromodeoxyuridine (5BrU)-substituted oligonucleotide with the sequence 5'-AGCGA5BrUATCGCT-3'. Cross-linking was to His62, mainly to the protein backbone. This observation provides further support for the mode of binding of GH5 to DNA proposed on the basis of the similarity between the X-ray crystal structure of GH5 and the DNA-bound structures of catabolite activator protein and hepatic nuclear factor 3 gamma [Ramakrishnan, V. (1994) Curr. Opin. Struct. Biol. 4. 44-50].

Subunit Structure of Rod cGMP-Phosphodiesterase

The rod cGMP phosphodiesterase (PDE) is the G-protein-activated effector enzyme that regulates the level of cGMP in vertebrate photoreceptor cells. Rod cGMP PDE is generally viewed as a heterotrimeric protein composed of catalytic alpha and beta subunits ( approximately90 kDa each) and two copies of the inhibitory subunit gamma ( approximately 10 kDa). However, the possibility that rod PDE could exist as distinct isoforms, such as alphaalphagamma2 and betabetagamma2 has not been ruled out. We have studied this question using cross-linking of PDE subunits with maleimidobenzoyl-N-hydroxysuccinimide ester and para-phenyldimaleimide. The cross-linking resulted in major products with molecular mass of 100 and 150 kDa, a doublet at approximately 180-190 kDa, and a doublet at approximately 210-220 kDa. Cross-linked products were analyzed using polyclonal-specific anti-PDEalphabeta, anti-PDEalpha, anti-PDEbeta, or anti-PDEgamma antibodies. The anti-PDEalpha and anti-PDEalphabeta antibodies recognized all the cross-linked products, whereas anti-PDEbeta and anti-PDEgamma antibodies did not interact with the 150-kDa band, indicating that the composition of this band is most likely alphaalpha. Similar analysis of cross-linked products of trypsin-treated PDE preparations revealed bands that are likely formed by PDEbeta subunit. The molecular size of holo-PDE and trypsin-activated PDE were studied using analytical ultracentrifugation in order to determine if oligomerization of PDE could account for the cross-linking of identical PDE subunits. The sedimentation analysis of both holo-PDE and ta-PDE revealed homogeneous samples with molecular masses of approximately220 and approximately150 kDa, respectively. These results indicate that PDE is likely a mixture of the major species alphabetagamma2, minor species alphaalphagamma2, and possibly betabetagamma2. Our data are consistent with the detection of low PDE activity in the rd mouse, which lacks any functional PDEbeta subunit.

Regulatory systems modulating the transcription of the pectinase genes of Erwinia chrysanthemi are conserved in Escherichia coli

To depolymerize plant pectin, the phytopathogenic enterobacterium Erwinia chrysanthemi produces five isoenzymes of pectate lyases encoded by the five genes pelA, pelB, pelC, pelD and pelE. In Er. chrysanthemi, all genes involved in pectin degradation are specifically controlled by the KdgR repressor and are induced in the presence of a pectin catabolic product, 2-keto-3-deoxygluconate (KDG). transcription of the pectinase genes is dependent on many environmental conditions. Transcriptional fusions present on low-copy-number plasmids were used to study the regulation of the pel genes in a heterologous host, Escherichia coli. Some physiological regulations that take place in Er. chrysanthemi are conserved in E. coli. The five pel fusions in E. coli are affected by growth phase, catabolite repression and anaerobic growth conditions and are induced in the presence of galacturonate, a sugar whose catabolism leads to the formation of KDG, the inducer of pel transcription in Er. chrysanthemi. Expression of pelE increased with the osmolarity of the culture medium. In contrast, the regulation of pel expression by temperature or nitrogen starvation, observed in Er. chrysanthemi, was not conserved in E. coli, suggesting that the mechanisms responsible for these regulations are specific to Er. chrysanthemi. Analysis of different E. coli mutants allowed some regulators affecting the transcription of the pel genes to be identified. In E. coli, the growth-phase regulation of the pel genes is not dependent on the RpoS sigma factor and the fnr gene is not involved in the increase of pel expression in oxygen-limited conditions. The gene hns, involved in the regulation of numerous genes, appears to affect pel expression but the effects of E. coli hns mutations are not related to osmoregulation. In contrast, this analysis clearly demonstrates the interchangeability of two regulatory systems of E. coli and Er. chrysanthemi: the global control exerted by the catabolite activator protein CAP and the specific regulation mediated by the KdgR repressor.

Uneven Distribution of GATC Motifs in the Escherichia coliChromosome, its Plasmids and its Phages

This work reconsiders the GATC motif distribution in a 1.6 Mb segment of the Escherichia coligenome, compared to its distribution in phages and plasmids. At first sight the distribution of GATC words looks random. But when a realistic model of the chromosome (made of average genes having the same codon usage as in the real chromosome), is used as a theoretical reference, strong biases are observed. GATC pairs such as GATCNNGATC are under-represented while there is a strong positive selection for motifs separated by 10, 19, 70 and 1100 bp. The last class is the only one present in E. coliparasites. It can be ascribed to the triggering sequences of the long-patch mismatch repair system. The 6 bp class overlaps with the consensus of CAP (catabolite activator protein) and FNR (fumarate/nitrate regulator) binding sites, thus accounting for counter-selection. The other classes, which could be targets for a nucleic acid binding protein, are almost always present inside protein coding sequences, and are members of clusters of GATC motifs. Analysis of the genes containing these motifs suggests that they correspond to a regulatory process monitoring the shift from anaerobic to aerobic growth conditions. In particular this regulation, closing down transcription of a large number of genes involved in intermediary metabolism would be well suited for the cold and oxygen shift from the mammal's gut to the standard environmental conditions. In this process the methylation status of GATC clusters would be very important for tuning transcription, and a DNA binding protein, probably a member of the cold-shock proteins family would be needed for alleviating the effects mediated by slackening of the pace of methylation during the shift.

Interactions between RNA polymerase and the positive and negative regulators of transcription at the Escherichia coli gal operon.

The simultaneous binding of Gal repressor (GalR), catabolite activator protein (CAP or CRP), and RNA polymerase (RNAP) to the promoter region of the Escherichia coli gal operon has been analyzed thermodynamically, by quantitative DNase I "footprint" titration analysis, and structurally, by the use of hydroxyl radical (.OH) and 5-phenylphenanthroline (5OPP) "footprinting." In the absence of regulatory proteins, the preference of RNAP for one (P1) of the two gal operon overlapping promoters (P1 and P2) is -0.4 +/- 0.2 kcal/mol, indicating only a small energetic preference for P1. The simultaneous binding of CAP and RNAP occurs with 10-fold cooperativity, with greater than 99% of the CAP-RNAP complex present at the P1 promoter. This cooperativity is inhibited by the binding of GalR to the upstream operator, OE, but does not result in the repartitioning of RNAP between the P1 and P2 promoters. These results suggest that the CAP-RNAP cooperativity and promoter partitioning are not linked and are consistent with a mechanism by which GalR binding to OE represses transcription by inhibiting the CAP-RNAP cooperativity. It is suggested that the CAP-RNAP cooperativity is dependent upon contacts made by the complex with the upstream DNA and that GalR binding to OE prevents these contacts from occurring. Changes in nuclease reactivity at the internal operator OI (centered at +53.5) take place upon RNAP binding. These changes are dependent on the DNA sequence present at OI and on the presence or absence of CAP. They are independent of the helical phasing between the promoters and OI and of the distance between them. These results suggest that RNAP can directly communicate with events occurring at both the external and the internal operator sequences without direct contact between repressor molecules bound at their cognate sites.

Cloning and transcription regulation of the ferric uptake regulatory gene of Campylobacter jejuni TGH9011

A Campylobacter jejuni (Cj) TGH9011 (ATCC 43431) gene homologous to the Escherichia coli ferric uptake regulatory gene (fur) has been cloned and characterized. Cj fur encodes a polypeptide consisting of 157 amino acids (aa) (18.1 kDa). The 5'-flanking region of the Cj fur gene contains two putative catabolite activator protein (CAP)-binding sequences and four Fur boxes or Fur-binding sequences ( FBS), implicating cAMP and autogenous regulation respectively. A major and a minor transcription start point ( tsp) were active in Fe(+) and Fe(-) media and three tsp were suppressed in Fe(+) condition. The major transcript has an unusually short leader sequence. The homology of the Cj Fur to other Proteobacteria Fur proteins is moderately low with identity ranging from 36.3% for Yersinia pestis to 31.8% for Legionella pneumophila. Multiple alignments of the Fur sequences identified three conserved motifs, I [aGLKvT1pR1KiL], II [eiG1ATvYR] and III [HHDH1vCldcGeviEf] (uppercase aa are identical in 12 or all 13 Fur sequences and lowercase aa are identical in six or more sequences). A truncated TGH9011 Fur missing 18 aa of the N terminus but retaining all three conserved motifs was shown to bind all four FBS sequences. The binding and transcription studies support autoregulation of fur expression in Cj.

Cloning and initial characterization of the Bordetella pertussis fur gene.

In several Gram-negative pathogens the fur (ferric uptake regulator) gene product controls the expression of many genes involved in iron uptake and virulence. To facilitate the study of iron-regulated gene expression in Bordetella pertussis, we cloned the fur gene from this organism. The B. pertussis fur gene product was 54% identical to the Escherichia coli Fur and complemented two E. coli fur mutants. As with the E. coli fur gene, sequences upstream of the B. pertussis fur were homologous to the consensus Fur-binding site and to the consensus catabolite activator protein binding site.

DNA-bend modulation in a repressor-to-activator switching mechanism.

Recent discoveries of activator proteins that distort DNA but bear no obvious activation domains have focused attention on the role of DNA structure in transcriptional regulation. Here we describe how the transcription factor MerR can mediate repression as well as activation through stereospecific modulation of DNA structure. The repressor form of MerR binds between the -10 and -35 promoter elements of the bacterial mercury-detoxification genes, PT, allowing RNA polymerase to form an inactive complex with PT and MerR at this stress-inducible promoter. Upon mercuric ion binding, Hg-MerR converts this polymerase complex into the transcriptionally active or 'open' form. We show here that MerR bends DNA towards itself in a manner similar to the bacterial catabolite-activator protein CAP, namely at two loci demarked by DNase I sensitivity, and that the activator conformation, Hg-MeR, relaxes these bends. This activator-induced unbending, when coupled with the previously described untwisting of the operator, remodels the promoter and makes it a better template for the poised polymerase.

Molecular interactions of 3',5'-cyclic purine analogues with the binding site of retinal rod ion channels.

Photoreceptor outer segments transduce information about incoming light levels through a class of ion channels that respond directly to changes in cytosolic 3',5'-cyclic guanosine monophosphate levels. A series of 3',5'-cyclic purine analogues with alterations at N1, C2, C6, or C8 positions was used to examine molecular interactions between the nucleotide and the channel. The maximal current activated by C2-altered analogues in excised membrane patches was less than the current activated by cGMP, and the K0.5, the concentration which activates 50% of the current in a patch, was increased. Nonpolar C8-substituted cAMP analogues activated more current than the parent cAMP with lower K0.5 values. This was in contrast to 8-amino-cAMP, which exhibited greatly reduced activity. The rank order of activity, based on K0.5 values, for C8-cAMP substituents was as follows: 8-azido- > 8-methylamino- > 8-benzylamino- > cAMP > 8-bromo- > 8-hydroxy- >> 8-amino-cAMP. 1,N6-Etheno-cAMP and N6-monobutyryl-cAMP activated a small fraction of the total possible current with high K0.5 values. Other analogues with alterations at N1 or C6 positions including N1-oxide-cAMP, 2-aminopurine riboside 3',5'-monophosphate, and N6-monosuccinyl-cAMP do not bind to the channel, suggesting that interactions with the channel in this region are essential for binding. In order to help interpret the changes in maximal current and K0.5 values compared to cGMP, molecular models of the active analogues were constructed and then docked into a molecular model of the cyclic nucleotide binding site of the retinal channel. This model, proposed by Kumar and Weber [(1992) Biochemistry 31, 4643-4649], was based on the crystal structure of cAMP bound to catabolite activator protein. Our modeling showed that the analogues were sterically accommodated within the binding site. No hydrogen bonds were predicted between the purine rings of cAMP and the pocket; however, Phe 533 on the beta 5 strand was predicted to form weak electrostatic interactions with C6 substituents on both cAMP and cGMP. The importance of contacts in this region of the binding pocket is further emphasized by the inactive analogues, all of which are altered at N1 or C6.

The functional subunit of a dimeric transcription activator protein depends on promoter architecture

In Class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase. In Class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 site. We have used an 'oriented heterodimers' approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase [CC(-61.5) and CC(-72.5)] and at one Class II promoter [CC(-41.5)]. Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter-proximal subunit of CAP. In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter-distal subunit of CAP.

Solution structure of the ets domain of Fli-1 when bound to DNA.

Members of the ets family of transcription factors share a conserved DNA-binding domain, the ets domain. By using multidimensional NMR, we have determined the structure of the ets domain of human Fli-1 in the DNA-bound form. It consists of three alpha-helices and a four-stranded beta-sheet, similar to structures of the class of helix-turn-helix DNA binding proteins first found in the catabolite activator protein of Escherichia coli. NMR and mutagenesis experiments suggest that in comparison to structurally related proteins, the ets domain uses a new variation of the helix-turn-helix motif for binding to DNA.

Secondary structure of the ETS domain places murine Ets-1 in the superfamily of winged helix-turn-helix DNA-binding proteins.

The members of the ets gene family of transcription factors are characterized by a conserved 85-residue DNA-binding region, termed the ETS domain, that lacks sequence homology to structurally characterized DNA-binding motifs. The secondary structure of the ETS domain of murine Ets-1 was determined on the basis of NMR chemical shifts, NOE and J-coupling constraints, amide hydrogen exchange, circular dichroism, and FT-IR spectroscopy. The ETS domain is composed of three alpha-helices (H) and four beta-strands (S) arranged in the order H1-S1-S2-H2-H3-S3-S4. The four-stranded antiparallel beta-sheet is the scaffold for a putative helix-turn-helix DNA recognition motif formed by helices 2 and 3. The 25 residues extending beyond the ETS domain to the native C-terminus of the truncated Ets-1 also contain a helical segment. On the basis of the similarity of this topology with that of catabolite activator protein (CAP), heat shock factor (HSF), and hepatocyte nuclear factor (HNF-3 gamma), we propose that ets proteins are members of the superfamily of winged helix-turn-helix DNA-binding proteins.