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The nine Pax transcription factors that constitute the mammalian family of paired domain (PD) factors play key roles in many developmental processes. As DNA binding transcription factors, they exhibit tremendous variability and complexity in their DNA recognition patterns. This is ascribed to the presence of multiple DNA binding structural domains, namely helix-turn-helix (HTH) domains. The PD contains two HTH subdomains and four of the nine Pax factors have an additional HTH domain, the homeodomain (HD). We now review these diverse DNA binding modalities together with their properties as transcriptional activators and repressors. The action of Pax factors on gene expression is also exerted through recruitment of chromatin remodelling complexes that introduce either activating or repressive chromatin marks. Interestingly, the recent demonstration that Pax7 has pioneer activity, the unique property to "open" chromatin, further underlines the mechanistic versatility and the developmental importance of these factors.

Homothorax belongs to the TALE-homeodomain family of transcription factors, together with its vertebrate counterparts, the Meis family of proto-oncogenes. It fulfills many important different functions during embryonic and larval developments in Drosophila, which encompass from subdivision and specification of body parts to assembly of heterochromatin structures. Hth interacts with Extradenticle, another member of the TALE-homeodomain family of conserved transcription factors, to facilitate its entrance to the nucleus. The many different functions described for Hth rely on the complexity of the locus, from which six different isoforms arise. The isoforms can be grouped into full-length and short versions, which contain either one or the two conserved domains of the protein (homeodomain and Exd-interacting domain). We have used molecular and genetic tools to analyze the levels of expression, the distribution and the function of the isoforms during embryonic development. Our results clearly show that the isoforms display distinct levels of expression and are differentially distributed in the embryo. This detailed study also shows that during normal embryonic development not all the Hth isoforms translocate Exd into the nucleus, suggesting that both the proteins can also function separately.We have demonstrated that the full-length Hth protein activates transcription of exd, augmenting the levels of exd mRNA in the cell. The higher levels of Exd protein in those cells facilitate its entrance to the nucleus.Our work demonstrates that hth is a complex gene that should not be considered as a functional unit. The roles of the different isoforms probably rely on their distinct protein domains and conformations and, at the end, on interactions with particular partners. © 2013 Elsevier Inc., Spanish MICINN BFU2009-12152; Fundación Ramón Areces; JAEPre011

The Vibrio fischer luxICDABEG genes are activated by autoinducer N-(3-oxohexanoyl)homoserine lactone and the LuxR protein. The LuxR contains 250 aa and consists of two domains. The C-domain, that extends from around residue 162 to the C-terminus, is thought to bind lux regulatory DNA and activate transcription of the luxICDABEG genes. The N-terminal domain, which binds the autoinducer, consists of about 70% residues of LuxR. In E. coli C-terminal domain can activate the lux genes in the absence of autoinducer. Previously it was shown that the ATP-dependent Lon protease of E. coli takes part in the negative regulation of the transcription of the V. fischeri lux operon and that LuxR is a target of Lon protease. Comparative analysis of effects of Lon protease on the V. fischeri luxICDABEG genes expression was made. Special constructed hybrid plasmids which permit the regulation of luxR, luxR 5'-deletion mutation were used and luICDABEG genes were activated independently and quantitatively. We show that the full length LuxR, but not C-terminal domain is a target protein for Lon protease. The transcription activity by full length LuxR protein isobserved when its intracellular concentration is about two order lower than that of its C-terminal domain.

Myeloid ecotropic insertion site (Meis)2 is a homeodomain protein containing a conserved homothorax (Hth) domain that is present in all Meis and Prep family proteins and in the Drosophila Hth protein. The Hth domain mediates interaction with Pbx homeodomain proteins, allowing for efficient DNA binding. Here we show that, like Meis1, Meis2 has a strong C-terminal transcriptional activation domain, which is required for full activation of transcription by homeodomain protein complexes composed of Meis2 and Pbx1. We also show that the activity of the activation domain is inhibited by the Hth domain, and that this autoinhibition can be partially relieved by the interaction of Pbx1 with the Hth domain of Meis2. Targeting of the Hth domain to DNA suggests that it is not a portable trans-acting repression domain. However, the Hth domain can inhibit a linked activation domain, and this inhibition is not limited to the Meis2 activation domain. Database searching reveals that the Meis3.2 splice variant, which is found in several vertebrate species, disrupts the Hth domain by removing 17 codons from the 5'-end of exon 6. We show that the equivalent deletion in Meis2 derepresses the C-terminal activation domain and weakens interaction with Pbx1. This work suggests that the transcriptional activity of all members of the Meis/Prep Hth protein family is subject to autoinhibition by their Hth domains, and that the Meis3.2 splice variant encodes a protein that bypasses this autoinhibitory effect.

Rice transcription factor RF2a binds to the BoxII cis element of the promoter of rice tungro bacilliform virus and activates promoter expression. The acidic acid-rich domain of RF2a is a transcription activator and has been partially characterized (Dai et al., 2003). The RF2a acidic domain (A; amino acids 49-116) was fused with the synthetic zinc finger ZF-TF 2C7 and was co-introduced with a reporter gene into transgenic Arabidopsis plants. Expression of the reporter gene was increased up to seven times by the effector. In transient assays in tobacco BY-2 protoplasts, we identified a subdomain comprising amino acids 56-84 (A5) that was equally as effective as an activator as the entire acidic domain. A chemically inducible system was used to show determined that A and A5 domains are equally as effective in transcription activation as the well-characterized VP16 activation domain. Bioinformatics analyses revealed that the A5 domain is present only in b-ZIP transcription factors. In dicots, the A domain contains an insertion of four amino acids that is not present in monocot proteins. The A5 domain, and similar domains in other b-ZIP transcription factors, is predicted to form an anti-parallel beta sheet structure.

SummaryProteins of the DUF199 family, present in all Gram-positive bacteria and best characterized by the WhiA sporulation control factor in Streptomyces coelicolor, are thought to act as genetic regulators. The crystal structure of the DUF199/WhiA protein from Thermatoga maritima demonstrates that these proteins possess a bipartite structure, in which a degenerate N-terminal LAGLIDADG homing endonuclease (LHE) scaffold is tethered to a C-terminal helix-turn-helix (HTH) domain. The LHE domain has lost those residues critical for metal binding and catalysis, and also displays an extensively altered DNA-binding surface as compared with homing endonucleases. The HTH domain most closely resembles related regions of several bacterial sigma70 factors that bind the −35 regions of bacterial promoters. The structure illustrates how an invasive element might be transformed during evolution into a larger assemblage of protein folds that can participate in the regulation of a complex biological pathway.

All seryl-tRNA synthetases (SerRSs) are functional homodimers with a C-terminal active site domain typical for class II aminoacyl-tRNA synthetases and an N-terminal domain involved in tRNA binding. The recently solved three-dimensional structure of Methanosarcina barkeri SerRS revealed the idiosyncratic features of methanogenic-type SerRSs; that is, an active site zinc ion, a unique tRNA binding domain, and an insertion of ∼30 residues in the catalytic domain, which adopt a helix-turn-helix (HTH) fold. Here, we present biochemical evidence for multiple roles of the HTH motif; it is important for dimerization of the enzyme, contributes to the overall stability, and is critical for the proper positioning of the tRNA binding domain relative to the catalytic domain. The changes in intrinsic fluorescence during denaturation of the wild-type M. barkeri SerRS and of the mutated variant lacking the HTH motif combined with cross-linking and gel analysis of protein subunits during various stages of the unfolding process revealed significantly reduced stability of the mutant dimers. In vitro kinetic analysis of enzymes, mutated in one of the N-terminal helices and the HTH motif, shows impaired tRNA binding and aminoacylation and emphasizes the importance of this domain for the overall architecture of the enzyme. The role of the idiosyncratic HTH motif in dimer stabilization and association between the catalytic and tRNA binding domain has been additionally confirmed by a yeast two-hybrid approach. Furthermore, we provide experimental evidence that tRNA binds across the dimer.

Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of ∼200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration.

NrpR is a transcriptional repressor of nitrogen assimilation genes that was recently discovered and characterized in the methanogenic archaeon Methanococcus maripaludis. NrpR homologues are widely distributed in Euryarchaeota and present in a few bacterial species. They exist in three different domain configurations: a single ORF encoding one NrpR domain following an N-terminal helix-turn-helix (HTH); a single ORF encoding two NrpR domains fused in tandem following an N-terminal HTH; and two separate ORFs, one with a single domain following an N-terminal HTH and one with a single domain without a HTH. Phylogenetic analysis indicated that the NrpR family forms five distinct groups: the single domain HTH type, the two domains of the double domain HTH type and the two separately encoded domains. To determine the function of diverse NrpR homologues, representative genes in were expressed an Methanococcus maripaludis nrpR deletion mutant. Homologues from species that possess a single gene restored regulated repression, regardless of domain structure. In the case of Methanosarcina acetivorans that contains two genes, both were required. The results show that distantly related NrpR homologues that are present in widely dispersed phyla regulate the expression of nitrogen assimilation genes in a similar fashion.

A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.

CheY-like phosphoacceptor (or receiver [REC]) domain is a common module in a variety of response regulators of the bacterial signal transduction systems. In this work, 4,610 response regulators, encoded in complete genomes of 200 bacterial and archaeal species, were identified and classified by their domain architectures. Previously uncharacterized output domains were analyzed and, in some cases, assigned to known domain families. Transcriptional regulators of the OmpR, NarL, and NtrC families were found to comprise almost 60% of all response regulators; transcriptional regulators with other DNA-binding domains (LytTR, AraC, Spo0A, Fis, YcbB, RpoE, and MerR) account for an additional 6%. The remaining one-third is represented by the stand-alone REC domain (∼14%) and its combinations with a variety of enzymatic (GGDEF, EAL, HD-GYP, CheB, CheC, PP2C, and HisK), RNA-binding (ANTAR and CsrA), protein- or ligand-binding (PAS, GAF, TPR, CAP_ED, and HPt) domains, or newly described domains of unknown function. The diversity of domain architectures and the abundance of alternative domain combinations suggest that fusions between the REC domain and various output domains is a widespread evolutionary mechanism that allows bacterial cells to regulate transcription, enzyme activity, and/or protein-protein interactions in response to environmental challenges. The complete list of response regulators encoded in each of the 200 analyzed genomes is available online at http://www.ncbi.nlm.nih.gov/Complete_Genomes/RRcensus.html .

The homothorax (hth) gene of Drosophila melanogaster is required for executing Hox functions, for head development, and for forming the proximodistal (PD) axis of the appendages. We show that alternative splicing of hth generates two types of protein isoforms, one that contains a DNA-binding homeodomain (HthFL) and one that does not contain a homeodomain (HDless). Both types of Hth isoforms include the evolutionarily conserved HM domain, which mediates a direct interaction with Extradenticle (Exd), another homeodomain protein. We show that although both HthFL and HDless isoforms of Hth can induce the nuclear localization of Exd, they carry out distinct sets of functions during development. Surprisingly, we find that many of hth’s functions, including PD patterning and most Hox-related activities, can be executed by the HDless isoforms. In contrast, antennal development shows an absolute dependency on the HthFL isoform. Thus, alternative splicing of hth results in the generation of multiple transcription factors that execute unique functions in vivo. We further demonstrate that the mouse ortholog of hth, Meis1, also encodes a HDless isoform, suggesting that homeodomain-less variants of this gene family are evolutionarily ancient.

Bacillus subtilis CodY protein is the best-studied member of a novel family of global transcriptional regulators found ubiquitously in low-G+C gram-positive bacteria. As for many DNA-binding proteins, CodY appears to have a helix-turn-helix (HTH) motif thought to be critical for interaction with DNA. This putative HTH motif was found to be highly conserved in the CodY homologs. Site-directed mutagenesis was used to identify amino acids within this motif that are important for DNA recognition and binding. The effects of each mutation on DNA binding in vitro and on the regulation of transcription in vivo from two target promoters were tested. Each of the mutations had similar effects on binding to the two promoters in vitro, but some mutations had differential effects in vivo.

Genome analysis has revealed that members of the Lrp family of transcriptional regulators are widely distributed among prokaryotes, both bacteria and archaea. The archetype Leucine-responsive Regulatory Protein from Escherichia coli is a global regulator involved in modulating a variety of metabolic functions, including the catabolism and anabolism of amino acids as well as pili synthesis. Most Lrp homologues, however, appear to act as specific regulators of amino acid metabolism-related genes. Like most prokaryotic transcriptional regulators, Lrp-like regulators consist of a DNA-binding domain and a ligand-binding domain. The crystal structure of the Pyrococcus furiosus LrpA revealed an N-terminal domain with a common helix-turn-helix fold, and a C-terminal domain with a typical alphabeta-sandwich fold. The latter regulatory domain constitutes a novel ligand-binding site and has been designated RAM. Database analysis reveals that the RAM domain is present in many prokaryotic genomes, potentially encoding (1) Lrp-homologues, when fused to a DNA-binding domain (2) enzymes, when fused as a potential regulatory domain to a catalytic domain, and (3) stand-alone RAM modules with unknown function. The architecture of Lrp regulators with two distinct domains that harbour the regulatory (effector-binding) site and the active (DNA-binding) site, and their separation by a flexible hinge region, suggests a general allosteric switch of Lrp-like regulators.

LuxR solo transcriptional regulators contain both an autoinducer binding domain (ABD; N-terminal) and a DNA binding Helix-Turn-Helix domain (HTH; C-terminal), but are not associated with a cognate N-acyl homoserine lactone (AHL) synthase coding gene in the same genome. Although a few LuxR solos have been characterized, their distributions as well as their role in bacterial signal perception and other processes are poorly understood. In this study we have carried out a systematic survey of distribution of all ABD containing LuxR transcriptional regulators (QS domain LuxRs) available in the InterPro database (IPR005143), and identified those lacking a cognate AHL synthase. These LuxR solos were then analyzed regarding their taxonomical distribution, predicted functions of neighboring genes and the presence of complete AHL-QS systems in the genomes that carry them. Our analyses reveal the presence of one or multiple predicted LuxR solos in many proteobacterial genomes carrying QS domain LuxRs, some of them harboring genes for one or more AHL-QS circuits. The presence of LuxR solos in bacteria occupying diverse environments suggests potential ecological functions for these proteins beyond AHL and interkingdom signaling. Based on gene context and the conservation levels of invariant amino acids of ABD, we have classified LuxR solos into functionally meaningful groups or putative orthologs. Surprisingly, putative LuxR solos were also found in a few non-proteobacterial genomes which are not known to carry AHL-QS systems. Multiple predicted LuxR solos in the same genome appeared to have different levels of conservation of invariant amino acid residues of ABD questioning their binding to AHLs. In summary, this study provides a detailed overview of distribution of LuxR solos and their probable roles in bacteria with genome sequence information.

A combination of sequence profile searching and structural protein analysis has revealed a novel type of small molecule binding domain that is involved in the allosteric regulation of prokaryotic amino acid metabolism. This domain, designated RAM, has been found to be fused to the DNA-binding domain of Lrp-like transcription regulators and to the catalytic domain of some metabolic enzymes, and has been found as a stand-alone module. Structural analysis of the RAM domain of Lrp reveals a betaalphabetabetaalphabeta-fold that is strikingly similar to that of the recently described ACT domain, a ubiquitous allosteric regulatory domain of many metabolic enzymes. However, structural alignment and re-evaluation of previous mutagenesis data suggest that the effector-binding sites of both modules are significantly different. By assuming that the RAM and ACT domains originated from a common ancestor, these observations suggest that their ligand-binding sites have evolved independently. Both domains appear to play analogous roles in controlling key steps in amino acid metabolism at the level of gene expression as well as enzyme activity.

Regulatory proteins in Escherichia coli with a helix-turn-helix (HTH) DNA binding motif show a position-function correlation such that repressors have this motif predominantly at the N terminus, whereas activators have the motif at the C-terminus extreme. Using this initial collection we identified by sequence comparison the exhaustive set of transcriptional regulators in 17 bacterial and 6 archaeal genomes. This enlarged set shows the same position-function correlation. The main question we address is whether this correlation is the result of common origin in evolution or the result of convergence. Evidence is presented supporting a common history at the origin of this correlation. We show the existence of a supergroup of eight repressor protein families sharing a conserved extended sequence comprising the classic HTH. Two of these repressor families (MarR and AsnC) originated before the divergence of Archaea and Bacteria. They are proposed at the origin of HTH-bearing transcriptional regulators currently present in Bacteria. The group of LysR proteins, with the HTH also at the N terminus, offers a control to the argument, since it shows clearly distinctive structural, functional, and evolutionary properties. This group of activator proteins, suggested to have originated within the Bacteria, has an advantageous gene organization to facilitate its horizontal transfer-used to conquer some Archaea-as well as negative autoregulation convenient for homeostasis, all of which agrees with this being the largest family in Bacteria. These results suggest that if shuffling of motifs occurred in Bacteria, it occurred only early in the history of these proteins, as opposed to what is observed in eukaryotic regulators.

Maize Dof1, one of the plant-specific Dof transcription factors, is involved in light-regulated gene expression. To elucidate the molecular mechanism underlying the activity of Dof1, in vivo functional analyses were carried out using transient expression assays with maize mesophyll protoplasts. The results suggest that the Dof domain alone, the region conserved among Dof factors, can mediate interaction with DNA in vivo and distinct Dof1 activities in greening and etiolated protoplasts. A region rich in basic amino acids was identified as a nuclear localization signal. Deletion analysis defined the transcriptional activation domain of 48 amino acids located in the C-terminus of Dof1. This activation domain was also found to be functional in both human cells and yeast, implying that Dof1 may stimulate transcription through a mechanism evolutionarily conserved among eukaryotes. A computer homology search with known transcription factors revealed that the activation domain of Dof1 displayed only a limited similarity to Activation domain II of animal transcription factor GATA-4. Mutational analysis revealed the critical role of a tryptophan residue within the activation domain of Dof1, as had been shown in Activation domain II of GATA-4.

Proteins that have a structure similar to those of LuxR and FixJ comprise a large subfamily of transcriptional activator proteins. Most members of the LuxR-FixJ family contain a similar amino-terminal receiver domain linked by a small region to a carboxy-terminal domain that contains an amino acid sequence similar to the helix-turn-helix (HTH) motif found in other DNA-binding proteins. GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family. Its 74-amino-acid sequence is similar over its entire length to the DNA binding region of this protein family, including the HTH motif. Therefore, GerE provides a simple model for studies of the role of this HTH domain in DNA binding. Toward this aim, we sought to identify the amino acids within this motif that are important for the specificity of binding to DNA. We examined the effects of single base pair substitutions in the high-affinity GerE binding site on the sigK promoter and found that nucleotides at positions +2, +3, and +4 relative to the transcription start site on the sigK promoter are important for a high-affinity interaction with GerE. We next examined the effects of single alanine substitutions at two positions in the HTH region of GerE on binding to wild-type or mutant target sites. We found that the substitution of an alanine for the threonine at position 42 of GerE produced a protein that binds with equal affinity to two sites that differ by 1 bp, whereas wild-type GerE binds with different affinities to these two sites. These results provide evidence that the amino acyl residues in or near the putative HTH region of GerE and potentially other members of the LuxR-FixJ family determine the specificity of DNA binding.

LuxR is the transcriptional activator for quorum-sensing control of luminescence in Vibrio fischeri . A series of alanine-scanning mutants spanning a predicted helix-turn-helix region in the DNA binding domain of LuxR was constructed, and the activity of each of the LuxR mutant proteins in recombinant Escherichia coli was investigated. The region covered by the mutagenesis spanned residues 190 to 224. About half of the alanine-scanning mutants showed activities similar to that of the wild-type LuxR: at least two were positive-control mutants, four appeared to be defective in DNA binding, and several others were characterized as DNA binding affinity mutants. This analysis, taken together with information about other bacterial transcription factors, provides insights into amino acid residues in LuxR that are involved in DNA binding and transcriptional activation.

Here we show that the C-terminal domain of LuxR activates the transcription of Aliivibrio fischeri luxICDABEG in Escherichia coli SKB178 gro + and E. coli OFB1111 groEL673 strains to the same level. Using affinity chromatography, we showed that GroEL binds to the N-terminal domain of LuxR, pointing to a GroEL/GroES requirement for the folding of the N-terminal domain of LuxR.

The bZIP proto-oncoprotein c-Fos activates transcription of a wide variety of genes involved in cell growth. The C-terminal activation domain of c-Fos is functionally independent of the remainder of the protein. Fos-AD corresponds to the C-terminal activation domain of human c-Fos (residues 216-380). Fos-AD suppresses (squelches) transcription in vitro, as expected for a functional activation domain lacking a DNA-binding domain. Fos-AD is unstructured and highly mobile, as demonstrated by circular dichroism spectra indicative of unfolded proteins, a lack of (1)H chemical shift dispersion, and negative (1)H-(15)N heteronuclear nuclear Overhauser effects. The hydrodynamic properties of Fos-AD are also consistent with an extended structure. We conclude that the C-terminal domain of human c-Fos is biologically active yet intrinsically disordered. Our results suggest that conformational disorder is an integral aspect of the diverse contributions to transcriptional regulation by c-Fos.

The Wilms' tumour suppressor protein WT1 contains a transcriptional regulatory domain that can either activate or repress transcription depending upon its cellular environment. The mechanistic basis for this dichotomy is unclear however. Here, we dissect the transcriptional regulatory domains of WT1. We find that a region within the domain of WT1 attributed to transcriptional repression is a potent suppressor of the activation domain at several promoters and in different cell types. In vitro transcription analysis suggests that the mechanism of suppression of the activation domain occurs at the level of transcription initiation. Furthermore we find that the WT1 suppression domain is able to inhibit a heterologous activation domain when fused in cis. Dissection of this domain resulted in the delineation of a 30 amino acid region that was sufficient to confer suppression of a transcriptional activation domain both in vivo and in vitro. Additionally, we find that the WT1 transcriptional activation domain interacts with the general transcription factor TFIIB and that this interaction is not affected by the suppression domain. Taken together, these studies suggest that the suppression domain of WT1 interacts with a cosuppressor protein to mediate inhibition of the WT1 transcriptional activation domain.

Metalloproteinase-disintegrins (ADAMs) are type 1 transmembrane proteins that contain a unique domain structure including a zinc-binding metalloproteinase domain. We have isolated cDNAs encoding two novel members of this family, ADAM29 and ADAM30 which show testis-specific expression. Three forms of ADAM29 were found that encode proteins of 820, 786 and 767 amino acids. All of the amino acid differences are located in the cytoplasmic domain. Two forms of ADAM30 were isolated that encode proteins of 790 and 781 amino acids, with the difference in the coding region occurring in the cytoplasmic domain. ADAM29 and ADAM30 map to human chromosome 4q34 and 1p11-13, respectively. An ancestral analysis of all known mammalian ADAMs indicates that the zinc-binding motif in the catalytic domain arose once in a common ancestor and was subsequently lost by those members lacking this motif.

The Drosophila PBC protein Extradenticle (Exd) is regulated at the level of its subcellular distribution: It is cytoplasmic in the absence of Homothorax (Hth), a Meis family member, and nuclear in the presence of Hth. Here we present evidence that, in the absence of Hth, Exd is exported from nuclei due to the activity of a nuclear export signal (NES). The activity of this NES is inhibited by the antibiotic Leptomycin B, suggesting that Exd is exported by a CRM1/exportin1-related export pathway. By analyzing the subcellular localization of Exd deletion mutants in imaginal discs and cultured cells, we identified three elements in Exd, a putative NES, a nuclear localization sequence (NLS), and a region required for Hth-mediated nuclear localization. This latter region coincides with a domain in Exd that binds Hth protein in vitro. When Exd is uncomplexed with Hth, the NES dominates over the NLS. When Exd is expressed together with Hth, or when the NES is deleted, Exd is nuclear. Thus, Hth is required to overcome the influence of the NES, possibly by inducing a conformational change in Exd. Finally, we provide evidence that Hth and Exd normally interact in the cytoplasm, and that Hth also has an NLS. We propose that in Exd there exists a balance between the activities of an NES and an NLS, and that Hth alters this balance in favor of the NLS.

MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix–turn–helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells. aMBF1 (archaeal MBF1) is very well conserved among most archaeal lineages; however, its function has so far remained elusive. To address this, we have conducted a molecular characterization of this aMBF1. Affinity purification of interacting proteins indicates that aMBF1 binds to ribosomal subunits. On sucrose density gradients, aMBF1 co-fractionates with free 30S ribosomal subunits as well as with 70S ribosomes engaged in translation. Binding of aMBF1 to ribosomes does not inhibit translation. Using NMR spectroscopy, we show that aMBF1 contains a long intrinsically disordered linker connecting the predicted N-terminal zinc-ribbon domain with the C-terminal HTH domain. The HTH domain, which is conserved in all archaeal and eukaryotic MBF1 homologues, is directly involved in the association of aMBF1 with ribosomes. The disordered linker of the ribosome-bound aMBF1 provides the N-terminal domain with high flexibility in the aMBF1–ribosome complex. Overall, our findings suggest a role for aMBF1 in the archaeal translation process.

The LIM domain protein rhombotin-2 (RBTN-2/TTG-2/LMO2) is involved in many processes, including leukemogenesis and erythropoiesis. It is thought that the principle role of RBTN-2 in these processes is to regulate transcription. To examine the potential for RBTN-2 to modulate transcription, we constructed RBTN-2/GAL4 DNA-binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct. From these studies we identified a transcription activation domain within the NH2 terminus of RBTN-2. This activation domain was further localized within a proline-rich 19-amino acid region. A second activation domain of 11 amino acids was also identified. This domain was located within the COOH terminus of RBTN-2, and functioned in mammalian cells but not in yeast. Furthermore, the two LIM domains of RBTN-2 were shown to function as transcription repression domains. Each individual LIM domain acted as an independent transcription repression domain on a heterologous activation domain. However, in context of full-length RBTN-2, the LIM domains selectively repressed the NH2-terminal activation domain, but had no effect on the COOH-terminal domain. Overall, these results demonstrate that the T-cell oncogene RBTN-2 is a complex transcription factor possessing multiple transcription regulatory modules, including two activation domains and two repression domains.

The helix-turn-helix (HTH) motif is commonly found in proteins that bind DNA, especially transcription factors. In recognizing DNA, the last helix of the HTH motif that is commonly referred to as the recognition helix inserts into the DNA major groove to mediate base-specific DNA recognition. The regulatory activity of transcription factors containing the HTH motif is dependent upon the context of the HTH domain within the larger protein and is found in both the basal transcriptional machinery and the gene- or operon-specific transcriptional activators and repressors. Importantly, however, the HTH motif also occurs in other protein families, especially multidomain proteins involved in nucleic acid metabolism, where it functions to recruit the protein to the nucleic acid. Finally, though much less common, some proteins instead utilize the HTH domain in protein–protein interactions.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., Extradenticle (Exd) and Homothorax (Hth) function as positive transcriptional cofactors of Hox proteins, helping them to bind specifically their direct targets. The posterior Hox protein Abdominal-B (Abd-B) does not require Exd/Hth to bind DNA; and, during embryogenesis, Abd-B represses hth and exd transcription. Here we show that this repression is necessary for Abd-B function, as maintained Exd/Hth expression results in transformations similar to those observed in loss-of-function Abd-B mutants. We characterize the cis regulatory module directly regulated by Abd-B in the empty spiracles gene and show that the Exd/Hth complex interferes with Abd-B binding to this enhancer. Our results suggest that this novel Exd/Hth function does not require the complex to bind DNA and may be mediated by direct Exd/Hth binding to the Abd-B homeodomain. Thus, in some instances, the main positive cofactor complex for anterior Hox proteins can act as a negative factor for the posterior Hox protein Abd-B. This antagonistic interaction uncovers an alternative way in which MEIS and PBC cofactors can modulate Abd-B like posterior Hox genes during development., This work was supported by the Programa Consolider, MICINN, European Regional Development Fund (FEDER), and Junta de Andalucía to JC-GH and by an EMBO short-term fellowship to MLR. YG was supported by the CNRS, Université de la Méditerranée, and grants from CEFIPRA, ANR, FRM, and ARC, and by a fellowship from MRT CEFIPRA to NS.

The Vibrio fischeri luminescence genes are activated by the LuxR protein and a diffusible signal termed the autoinducer. LuxR consists of two domains, a C-terminal transcriptional activator domain, and an N-terminal autoinducer-binding domain, which serves to regulate the function of the C-terminal domain. We have isolated and characterized an intragenic suppressor of a mutation that maps to the N-terminal domain and blocks autoinducer binding. The suppressor changes an alanine residue at position-221 in the C-terminal domain to a valine. In Escherichia coli, the suppressor allows partial activation of the V. fischeri luminescence genes although E. coli containing this protein remains unable to bind autoinducer. To further analyze the influence of the second-site mutation on luxR function, we constructed a luxR gene that coded for a protein with a wild-type N-terminal domain and with the ala-221 to val substitution in the C-terminal domain. This protein activated the luminescence genes in the presence or absence of autoinducer, and it bound autoinducer at levels comparable to the wild-type LuxR protein. Apparently, the alanine to valine substitution at position-221 allows activity of the C-terminal domain in a fashion independent of whether autoinducer is bound to the N-terminal domain.

Light-oxygen-voltage (LOV) domains serve as the photosensory modules for a wide range of plant and bacterial proteins, conferring blue light-dependent regulation to effector activities as diverse as enzymes and DNA binding. LOV domains can also be engineered into a variety of exogenous targets, allowing similar regulation for new protein-based reagents. Common to these proteins is the ability for LOV domains to reversibly form a photochemical adduct between an internal flavin chromophore and the surrounding protein, using this to trigger conformational changes that affect output activity. Using the Erythrobacter litoralis protein EL222 model system that links LOV regulation to a helix-turn-helix (HTH) DNA binding domain, we demonstrated that the LOV domain binds and inhibits the HTH domain in the dark, releasing these interactions upon illumination [Nash, A. I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 9449-9454]. Here we combine genomic and in vitro selection approaches to identify optimal DNA binding sites for EL222. Within the bacterial host, we observe binding at several genomic sites using a 12 bp sequence consensus that is also found by in vitro selection methods. Sequence-specific alterations in the DNA consensus reduce EL222 binding affinity in a manner consistent with the expected binding mode, a protein dimer binding to two repeats. Finally, we demonstrate the light-dependent activation of transcription of two genes adjacent to an EL222 binding site. Taken together, these results shed light on the native function of EL222 and provide useful reagents for further basic and applications research of this versatile protein.

Background Multiprotein-bridging factor 1 (MBF1) is a transcriptional co-activator that bridges a sequence-specific activator (basic-leucine zipper (bZIP) like proteins (e.g. Gcn4 in yeast) or steroid/nuclear-hormone receptor family (e.g. FTZ-F1 in insect)) and the TATA-box binding protein (TBP) in Eukaryotes. MBF1 is absent in Bacteria, but is well- conserved in Eukaryotes and Archaea and harbors a C-terminal Cro-like Helix Turn Helix (HTH) domain, which is the only highly conserved, classical HTH domain that is vertically inherited in all Eukaryotes and Archaea. The main structural difference between archaeal MBF1 (aMBF1) and eukaryotic MBF1 is the presence of a Zn ribbon motif in aMBF1. In addition MBF1 interacting activators are absent in the archaeal domain. To study the function and therefore the evolutionary conservation of MBF1 and its single domains complementation studies in yeast (mbf1Δ) as well as domain swap experiments between aMBF1 and yMbf1 were performed. Results In contrast to previous reports for eukaryotic MBF1 (i.e. Arabidopsis thaliana, insect and human) the two archaeal MBF1 orthologs, TMBF1 from the hyperthermophile Thermoproteus tenax and MMBF1 from the mesophile Methanosarcina mazei were not functional for complementation of an Saccharomyces cerevisiae mutant lacking Mbf1 (mbf1Δ). Of twelve chimeric proteins representing different combinations of the N-terminal, core domain, and the C-terminal extension from yeast and aMBF1, only the chimeric MBF1 comprising the yeast N-terminal and core domain fused to the archaeal C-terminal part was able to restore full wild-type activity of MBF1. However, as reported previously for Bombyx mori, the C-terminal part of yeast Mbf1 was shown to be not essential for function. In addition phylogenetic analyses revealed a common distribution of MBF1 in all Archaea with available genome sequence, except of two of the three Thaumarchaeota; Cenarchaeum symbiosum A and Nitrosopumilus maritimus SCM1. Conclusions The absence of MBF1-interacting activators in the archaeal domain, the presence of a Zn ribbon motif in the divergent N-terminal domain of aMBF1 and the complementation experiments using archaeal- yeast chimeric proteins presented here suggests that archaeal MBF1 is not able to functionally interact with the transcription machinery and/or Gcn4 of S. cerevisiae. Based on modeling and structural prediction it is tempting to speculate that aMBF1 might act as a single regulator or non-essential transcription factor, which directly interacts with DNA via the positive charged linker or the basal transcription machinery via its Zn ribbon motif and the HTH domain. However, also alternative functions in ribosome biosynthesis and/or functionality have been discussed and therefore further experiments are required to unravel the function of MBF1 in Archaea. Reviewers This article was reviewed by William Martin, Patrick Forterre, John van der Oost and Fabian Blombach (nominated by Eugene V Koonin (United States)). For the full reviews, please go to the Reviewer's Reports section.

The I-mfa domain proteins I-mfa and HIC are considered to be candidate tumor suppressor genes and have been shown to be involved in transcriptional regulation. We show here that I-mfa and HIC specifically interact with SEI-1 through their C-terminal I-mfa domains in vivo. This interaction affects the intracellular localization of I-mfa and requires the region of SEI-1 between 30 and 90 amino acids, which includes its SERTA domain, and results in repression of its intrinsic transcriptional activity. I-mfa also decreases the levels of the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA, the expression of which is coregulated by E2F·DP-1 and SEI-1 in an interaction-dependent manner in vitro. In addition, I-mfa also specifically interacts with other SERTA domain-containing proteins, including SEI-2, SEI-3, SERTAD3 and SERTAD4, through its I-mfa domain in vivo. This interaction also affects the intracellular localization of I-mfa and represses the intrinsic transcriptional activities of SEI-2 and SERTAD3, which are also involved in the E2F-dependent transcription. These data reveal for the first time that I-mfa domain proteins interact with SERTA domain proteins and negatively regulate their transcriptional activity. Because SEI-1, SEI-2 and SERTAD3, whose intrinsic transcriptional activities are repressed by I-mfa, are suggested to be oncogenes, I-mfa domain proteins may be involved in their oncogenic functions by negatively regulating their transcriptional activities.

Bacteria can modulate their behavior by releasing and responding to the accumulation of signal molecules. This population co-ordination, referred to as quorum sensing, is prevalent in Gram-negative and Gram-positive bacteria. The essential constituents of quorum-sensing systems include a signal producer, or synthase, and a cognate transcriptional regulator that responds to the accumulated signal molecules. With the availability of bacterial genome sequences and an increased elucidation of quorum-sensing circuits, genes that code for additional transcriptional regulators, usually in excess of the synthase, have been identified. These additional regulators are referred to as 'orphan' regulators, because they are not directly associated with a synthase. Here, we review orphan regulators characterized in various Gram-negative bacteria and their role in expanding the bacterial regulatory network.

One of the most abundant and widely distributed groups of Helix-turn-helix (HTH) transcription factors is the metabolite-responsive GntR family of regulators (>8500 members in the Pfam database; Jan 2009). These proteins contain a DNA-binding HTH domain at the N terminus of the protein and an effector-binding and/or oligomerisation domain at the C terminus, where upon on binding an effector molecule, a conformational change occurs in the protein which influences the DNA-binding properties of the regulator resulting in repression or activation of transcription. This review summarises what we know about the distribution, structure, function and classification of these regulators and suggests that they may have a future role in biotechnology.

Ectromelia virus (ECTV) encodes an IFN-γ-binding protein (IFN-γBP ECTV ) that disrupts IFN-γ signaling and its ability to induce an antiviral state within cells. IFN-γBP ECTV is an important virulence factor that is highly conserved (>90%) in all orthopoxviruses, including variola virus, the causative agent of smallpox. The 2.2-Å crystal structure of the IFN-γBP ECTV /IFN-γ complex reveals IFN-γBP ECTV consists of an IFN-γR1 ligand-binding domain and a 57-aa helix-turn-helix (HTH) motif that is structurally related to the transcription factor TFIIA. The HTH motif forms a tetramerization domain that results in an IFN-γBP ECTV /IFN-γ complex containing four IFN-γBP ECTV chains and two IFN-γ dimers. The structure, combined with biochemical and cell-based assays, demonstrates that IFN-γBP ECTV tetramers are required for efficient IFN-γ antagonism.

Pax-8 is a transcription factor belonging to the PAX genes superfamily and its crucial role has been proven both in embryo and in the adult organism. Pax-8 activity is regulated via a redoxbased mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys45 and Cys57). These residues belong to a highly evolutionary conserved DNA binding site: the Paired Box (Prd) domain. Crystallographic protein-DNA complexes of the homologues Pax-6 and Pax-5 showed a bipartite Prd domain consisting of two helix-turn-helix (HTH) motifs separated by an extended linker region. Here, by means of nuclear magnetic resonance, we show for the first time that the HTH motifs are largely defined in the unbound Pax-8 Prd domain. Our findings contrast with previous induced fit models, in which Pax-8 is supposed to largely fold upon DNA binding. Importantly, our data provide the structural basis for the enhanced chemical reactivity of residues Cys45 and Cys57 and explain clinical missense mutations that are not obviously related to the DNA binding interface of the paired box domain. Finally, sequence conservation suggests that our findings could be a general feature of the Pax family transcription factors.

Among the transcription factors, the helix-turn-helix (HTH) GntR family comprised of FadR, HutC, MocR, YtrA, AraR, and PlmA subfamilies regulates the most varied biological processes. Generally, proteins belonging to this family contain an N-terminal DNA-binding domain and a C-terminal effector-binding/oligomerization domain. The members of the YtrA subfamily are much shorter than other members of this family, with chain lengths of 120-130 residues with about 50 residues located in the C-terminal domain. Because of this length, the mode of dimerization and the ability to bind effectors by the C-terminal domain are puzzling. Here, we first report the structure of the transcription factor CGL2947 from Corynebacterium glutamicum, which belongs to the YtrA family. The monomer is composed of a DNA-binding domain containing a winged HTH motif in the N terminus and two helices (alpha4 and alpha5) with a fishhook-shaped arrangement in the C terminus. Helices alpha4 and alpha5 of two monomers intertwine together to form a novel homodimer assembly. The effector-accommodating pocket with 2-methyl-2,4-pentanediol (MPD) docked was located, and it was suggested to represent a novel mode of effector binding. The structures in two crystal forms (MPD-free and -bound in the proposed effector-binding pocket) were solved. The structural variations have implications regarding how the effector-induced conformational change modulates DNA affinity for YtrA family members.

The New York Structural GenomiX Research Consortium (NYSGXRC) has selected the protein coded by yxaF gene from Bacillus subtilis as a target for structure determination. The yxaF protein has 191 residues with a molecular mass of 21 kDa and had no sequence homology to any structure in the Protein Data Bank (PDB) at the time of target selection. We aimed to elucidate the three-dimensional structure for the putative protein yxaF to better understand the relationship between protein sequence, structure, and function. This protein is annotated as a putative helix-turn-helix (HTH) type transcriptional regulator. Many transcriptional regulators like TetR and QacR use a structurally well-defined DNA-binding HTH motif to recognize the target DNA sequences. DNA-HTH motif interactions have been extensively studied. As the HTH motif is structurally conserved in many regulatory proteins, these DNA-protein complexes show some similarity in DNA recognition patterns. Many such regulatory proteins have a ligand-binding domain in addition to the DNA-binding domain. Structural studies on ligand-binding regulatory proteins provide a wealth of information on ligand-, and possibly drug-, binding mechanisms. Understanding the ligand-binding mechanism may help overcome problems with drug resistance, which represent increasing challenges in medicine. The protein encoded by yxaF, hereafter called T1414, shows foldmore » similar to QacR repressor and TetR/CamR repressor and possesses putative DNA and ligand-binding domains. Here, we report the crystal structure of T1414 and compare it with structurally similar drug and DNA-binding proteins.« less

Dof (DNA-binding with one finger) domain proteins are plant-specific transcription factors with a highly conserved DNA-binding domain, which presumably includes a single C(2)-C(2) zinc finger. During the past decade, numerous Dof domain proteins have been identified in both monocots and dicots including maize, barley, wheat, rice, tobacco, Arabidopsis, pumpkin, potato, and pea. Biochemical, molecular biological and molecular genetic analyses revealed that Dof domain proteins function as a transcriptional activator or a repressor involved in diverse plant-specific biological processes. Although more physiological roles of Dof domain proteins would be elucidated in future because of numerous Dof domain proteins in plants, it is already evident that the Dof domain proteins play critical roles as transcriptional regulators in plant growth and development. Here I summarize our current knowledge about Dof domain proteins.

The transposase of IS911, a member of the IS3 family of bacterial insertion sequences, is composed of a catalytic domain located at its C-terminal end and a DNA binding domain located at its N-terminal end. Analysis of the transposases of over 60 members of the IS3 family revealed the presence of a helix-turn-helix (HTH) motif within the N-terminal region. Alignment of these potential secondary structures further revealed a completely conserved tryptophan residue similar to that found in the HTH motifs of certain homeodomain proteins. The analysis also uncovered a similarity between the IS3 family HTH and that of members of the LysR family of bacterial transcription factors. This information was used to design site-directed mutations permitting an assessment of its role in transposase function. A series of in vivo and in vitro tests demonstrated that the HTH domain is important in directing the transposase to bind the terminal inverted repeats of IS911.

The genes coding for the response regulators ARR1 and ARR2 have previously been identified by in silico screening of an expression sequence tag database and subsequent cloning from both Arabidopsis cDNA and genomic libraries. Their structures, in which the N-terminal signal receiver domain is followed by the output domain, are characteristic of typical bacterial response regulators of the two-component regulatory systems that control responses to a variety of environmental stimuli. Here we present evidence that these response regulators actually work as transcription factors. ARR1 and ARR2 were localized in the nuclei of plant cells regardless of the presence or absence of their signal receiver domain. Their middle segments, which faintly resemble the mammalian oncogene product Myb, were capable of binding double-stranded DNA in a sequence-specific manner in vitro. Their C-terminal halves functioned as transactivation domains in plant cells when combined with the DNA-binding domain of yeast GAL4. They thus possess all the essential components of a transcriptional activator. Both ARR1 and ARR2 promoted expression of a reporter gene in plant cells through their own target sequence. Truncation of their N-terminal signal receiver domain led to an increase in transactivation. An as yet unidentified phospho-relay signal may modulate the capability for transactivation and/or DNA binding through the signal receiver domain.

Many examples of enzymes that have lost their catalytic activity and perform other biological functions are known. The opposite situation is rare. A previously unnoticed structural similarity between the lambda integrase family (Int) proteins and the AraC family of transcriptional activators implies that the Int family evolved by duplication of an ancient DNA-binding homeodomain-like module, which acquired enzymatic activity. The two helix-turn-helix (HTH) motifs in Int proteins incorporate catalytic residues and participate in DNA binding. The active site of Int proteins, which include the type IB topoisomerases, is formed at the domain interface and the catalytic tyrosine residue is located in the second helix of the C-terminal HTH motif. Structural analysis of other 'tyrosine' DNA-breaking/rejoining enzymes with similar enzyme mechanisms, namely prokaryotic topoisomerase I, topoisomerase II and archaeal topoisomerase VI, reveals that the catalytic tyrosine is placed in a HTH domain as well. Surprisingly, the location of this tyrosine residue in the structure is not conserved, suggesting independent, parallel evolution leading to the same catalytic function by homologous HTH domains. The 'tyrosine' recombinases give a rare example of enzymes that evolved from ancient DNA-binding modules and present a unique case for homologous enzymatic domains with similar catalytic mechanisms but different locations of catalytic residues, which are placed at non-homologous sites.

Likely DNA-binding domains in archaeal proteins were analyzed using sequence profile methods and available structural information. It is shown that all archaea encode a large number of proteins containing the helix-turn-helix (HTH) DNA-binding domains whose sequences are much more similar to bacterial HTH domains than to eukaryotic ones, such as the PAIRED, POU and homeodomains. The predominant class of HTH domains in archaea is the winged-HTH domain. The number and diversity of HTH domains in archaea is comparable to that seen in bacteria. The HTH domain in archaea combines with a variety of other domains that include replication system components, such as MCM proteins, translation system components, such as the alpha-subunit of phenyl-alanyl-tRNA synthetase, and several metabolic enzymes. The majority of the archaeal HTH-containing proteins are predicted to be gene/operon-specific transcriptional regulators. This apparent bacterial-type mode of transcription regulation is in sharp contrast to the eukaryote-like layout of the core transcription machinery in the archaea. In addition to the predicted bacterial-type transcriptional regulators, the HTH domain is conserved in archaeal and eukaryotic core transcription factors, such as TFIIB, TFIIE-alpha and MBF1. MBF1 is the only highly conserved, classical HTH domain that is vertically inherited in all archaea and eukaryotes. In contrast, while eukaryotic TFIIB and TFIIE-alpha possess forms of the HTH domain that are divergent in sequence, their archaeal counterparts contain typical HTH domains. It is shown that, besides the HTH domain, archaea encode unexpectedly large numbers of two other predicted DNA-binding domains, namely the Arc/MetJ domain and the Zn-ribbon. The core transcription regulators in archaea and eukaryotes (TFIIB/TFB, TFIIE-alpha and MBF1) and in bacteria (the sigma factors) share no similarity beyond the presence of distinct HTH domains. Thus HTH domains might have been independently recruited for a role in transcription regulation in the bacterial and archaeal/eukaryotic lineages. During subsequent evolution, the similarity between archaeal and bacterial gene/operon transcriptional regulators might have been established and maintained through multiple horizontal gene transfer events.

We show that homothorax (hth) is required for the Hox genes to pattern the body of the fruit fly, Drosophila melanogaster. hth is necessary for the nuclear localization of an essential HOX cofactor, Extradenticle (EXD), and encodes a homeodomain protein that shares extensive identity with the product of Meis1, a murine proto-oncogene. MEIS1 is able to rescue hth mutant phenotypes and can induce the cytoplasmic-to-nuclear translocation of EXD in cell culture and Drosophila embryos. Thus, Meis1 is a murine homolog of hth. MEIS1/HTH also specifically binds to EXD with high affinity in vitro. These data suggest a novel and evolutionarily conserved mechanism for regulating HOX activity in which a direct protein–protein interaction between EXD and HTH results in EXD's nuclear translocation.

In Escherichia coli, subdomains 2.4 and 4.2 of the primary transcription factor sigma 70 are the most highly conserved regions and are responsible for the recognition of -10 and -35 promoter elements respectively. Mutational studies provide evidence to this end and indicate that the side chains of subdomain 4.2 make specific contacts with the nucleotides at -35. Subdomain 4.2 is highly conserved among group-1 sigma factors and is strongly homologous to the classical helix-turn-helix (HTH) motif shared by bacteriophage lembda cl, Cro, the CAP protein and other homeodomain proteins, suggesting that sigma factor also belongs to the HTH class of proteins. In this study, a single point mutation of the conserved hydrophobic residue valine at position 576, in the 4.2 subdomain results in a mutant that is transcriptionally inefficient although conformationally similar to wild-type sigma. The mutant sigma, like wild-type, migrates as a 87 kDa protein on SDS gels and has 50% helicity. However, transcription at "extended -10 promoter' by RNA polymerase containing mutant sigma 70-V576G, synthesized appreciable amount of RNA product, when compared with that generated by sigma 70-W434G, a mutation in -10 DNA binding domain. A model of HTH motif for the conserved 20 residue region of 4.2 domain of E. coli sigma 70 as well as its mutant sigma 70-V576G and sigma 70-V576T were constructed based on five other homologous HTH motifs from DNA-protein complexes for which X-ray or NMR structure is available. A B-DNA structure was designed for -35 region using sequence dependent base pair parameters. The modeled HTH structure was docked into the major groove formed by the -35 hexamer DNA using the DNA-recognition rules and amino acid-nucleotide base contact information of homologous DNA-protein complexes. Analysis of the residue contact information of the model was tested and found to have good agreement with the experimental reports.

The Vibrio fischeri luminescence genes are regulated by the LuxR protein and an N-acyl homoserine lactone compound termed the autoinducer. The C-terminal one-third of LuxR contains a domain that can interact with the transcription complex and activate the luminescence genes. On the basis of limited evidence it has been suggested that the N-terminal two-thirds of LuxR constitutes a domain that serves to bind the autoinducer. We show that tritium-labeled autoinducer binds to Escherichia coli cells in which LuxR is overexpressed. We also show that tritium-labeled autoinducer binds to E. coli in which truncated LuxR proteins missing portions of the C-terminal domain are expressed but does not bind to E. coli cells in which truncated LuxR proteins missing portions of the N-terminal region are expressed. Our results provide evidence that the autoinducer binds to LuxR and that in E. coli the N-terminal two-thirds of LuxR can fold into a polypeptide capable of binding the autoinducer in the absence of the C-terminal domain.