SMART MODE:

NORMAL GENOMIC

• Egland KA, Greenberg EP
• Quorum sensing in Vibrio fischeri: analysis of the LuxR DNA binding region by alanine-scanning mutagenesis.
• J Bacteriol. 2001; 183: 382-6
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• 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.

• Perez-Rueda E, Collado-Vides J
• Common history at the origin of the position-function correlation in transcriptional regulators in archaea and bacteria.
• J Mol Evol. 2001; 53: 172-9
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• 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.

• Griffith KL, Wolf RE Jr
• Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation.
• Mol Microbiol. 2001; 40: 1141-54
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• SoxS is the direct transcriptional activator of at least 15 genes of the Escherichia coli superoxide regulon. SoxS is small (107 amino acids), binds DNA as a monomer and recognizes a highly degenerate DNA binding site, termed 'soxbox'. Like other members of the AraC/XylS family, SoxS has two putative helix-turn-helix (HTH) DNA-binding motifs, and it has been proposed that each HTH motif recognizes a highly conserved recognition element of the soxbox. To determine which nucleotides are important for SoxS binding, we conducted a systematic mutagenesis of the DNA binding sites for SoxS in the zwf and fpr promoters and determined the effect of the soxbox mutations on SoxS DNA binding and transcription activation in vivo by measuring beta-galactosidase activity in strains with fusions to lacZ. We found that the sequences GCAC and CAAA, termed recognition elements 1 and 2 (RE 1 and RE 2), respectively, are critical for SoxS binding, as mutations within these elements severely hinder or eliminate SoxS-dependent transcription activation; substitutions within RE 2 (CAAA), however, are tolerated better than changes within RE 1 (GCAC). Although substitutions at the seven positions separating the two REs had only a modest effect on SoxS binding, AT basepairs were favoured within this 'spacer' region, presumably because, by facilitating DNA bending, they help bring the two recognition elements into proper juxtaposition. We also found that the 'invariant A' present at position 1 of 14/15 functional soxboxes identified thus far is important for SoxS binding, as a change to any other nucleotide at this position reduced SoxS-dependent transcription by approximately 50%. In addition, positions surrounding the REs seem to show a context effect, in that certain substitutions there have little or no effect when the RE has the optimal binding sequence, but produce a pronounced effect when the RE has a suboptimal sequence. We propose that these nucleotides play an important role in effecting differential expression from the various promoters. Lastly, we used gel retardation assays to show that alterations in transcription activation in vivo are caused by effects on DNA binding. Based on this exhaustive mutagenesis, we propose the following optimal sequence for SoxS binding: AnVGCACWWWnKRHCAAAHn (n = A, C, G, T; V = A, C, G; W = A, T; K = G, T; R = A, G; H = A, C, T).

• Harmer T, Wu M, Schleif R
• The role of rigidity in DNA looping-unlooping by AraC.
• Proc Natl Acad Sci U S A. 2001; 98: 427-31
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• We applied two experiments useful in the study of ligand-regulated DNA binding proteins to AraC, the dimeric regulator of the Escherichia coli l-arabinose operon. In the absence of arabinose, AraC prefers to loop DNA by binding to two half-sites that are separated by 210 base pairs, and in the presence of arabinose it prefers to bind to adjacently located half-sites. The basis for this ligand-regulated shift in binding appears to result from a shift in the rigidity of the system, where rigidity both in AraC protein in the absence of arabinose, and in the DNA are required to generate the free energy differences that produce the binding preferences. Eliminating the dimerization domains and connecting the two DNA binding domains of AraC by a flexible peptide linker should provide a protein whose behavior mimics that of AraC when there is no interaction between its dimerization and DNA binding domains. The resulting protein bound to adjacent half-sites on the DNA, like AraC protein in the presence of arabinose. When the two double-stranded DNA half-sites were connected by 24 bases of single-stranded, flexible DNA, wild-type AraC protein bound to the DNA in the presence and absence of arabinose with equal affinity, showing that AraC modulates its DNA binding affinity in response to arabinose by shifting the relative positions of its DNA binding domains. These results are consistent with the light switch mechanism for the action of AraC, refine the model, and extend the range of experimental tests to which it has been subjected.

• Nicholas HB Jr, Arst HN Jr, Caddick MX
• Evaluating low level sequence identities. Are Aspergillus QUTA and AROM homologous?
• Eur J Biochem. 2001; 268: 414-9
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• A review published several years ago [Hawkins, A.R. & Lamb, H.K. (1995) Eur. J. Biochem. 232, 7-18] proposed that genetic, biochemical and physiological data can override sequence comparison in the determination of homology in instances where structural information is unavailable. Their lead example was the hypothesis that the transcriptional activator protein for quinate catabolism in Aspergillus nidulans, QUTA, is derived from the pentafunctional AROM protein by a gene duplication followed by cleavage [Hawkins, A.R., Lamb, H.K., Moore, J.D. & Roberts, C.F. (1993) Gene 136, 49-54]. We tested this hypothesis by a sensitive combination of position-specific log-odds scoring matrix methods. The position-specific log-odds scoring matrices were derived from a large number of 3-dehydroquinate synthase and 5-enolpyruvylshikimate-3-phosphate synthase domains that were proposed to be the domains from the AROM protein that gave rise to the transcriptional activator protein for quinate metabolism. We show that the degree and pattern of similarity between these position-specific log-odds scoring matrices and the transcriptional activator protein for quinate catabolism in A. nidulans is that expected for random sequences of the same composition. This level of similarity provides no support for the suggested gene duplication and cleavage. The lack of any trace of evidence for homology following a comprehensive sequence analysis indicates that the homology hypothesis is without foundation, underlining the necessity to accept only similarity of sequence and/or structure as evidence of evolutionary relatedness. Further, QUTA is homologous throughout its entire length to an extended family of fungal transcriptional regulatory proteins, rendering the hypothesized QUTA-AROM homology even more problematic.

• Steinmetzer K, Hillisch A, Behlke J, Brantl S
• Transcriptional repressor CopR: amino acids involved in forming the dimeric interface.
• Proteins. 2000; 39: 408-16
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• Plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. It acts as a transcriptional repressor at the essential repR promoter. Furthermore, CopR prevents convergent transcription from the repR and the antisense promoter, thereby indirectly increasing the amount of antisense-RNA, the second regulatory component. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'CGTG. Previously, a CopR structural model was built and used to identify amino acids involved in DNA binding. These data showed that CopR is a HTH protein belonging to the lambda repressor superfamily and allowed the identification of two amino acids involved in specific DNA recognition. Here, we describe site-directed mutagenesis in combination with EMSA, dimerization studies using sedimentation equilibrium, and CD measurements to verify the model predictions concerning amino acids involved in dimerization. With this approach, the dimeric interface could be located between amino acids I44 and L62. F5 located at the N-terminus is additionally required for proper folding, and could, therefore, not be unequivocally assigned to the dimeric interface. CD measurements at protein concentrations well below K(Dimer) revealed that the monomer of CopR is folded.

• Gillette WK, Martin RG, Rosner JL
• Probing the Escherichia coli transcriptional activator MarA using alanine-scanning mutagenesis: residues important for DNA binding and activation.
• J Mol Biol. 2000; 299: 1245-55
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• The MarA transcriptional activator binds to a 20 bp asymmetric degenerate sequence (marbox) located at different positions and orientations within the promoters of the genes of the Escherichia coli mar regulon. Solution of the MarA-marbox X-ray crystallographic structure suggested the presence of base-specific and non-specific interactions between the marbox and two helix-turn-helix (HTH) motifs on the monomeric MarA. Here, we use alanine-scanning mutagenesis and DNA retardation analysis to: (i) evaluate the contacts between MarA and the marboxes of five differently configured mar regulon promoters; (ii) assess the role of conserved hydrophobic amino acid residues for MarA activity; and (iii) identify residues required for RNA polymerase activation. These analyses revealed that the phosphate-backbone contacts and hydrogen bonds with the bases of the marbox are more significant for DNA binding than are the van der Waals interactions. While both N and C-terminal HTH motifs make essential contributions to binding site affinity, MarA is more sensitive to alterations in the N-terminal HTH. In a similar way, the activity of MarA is more sensitive to alterations in the hydrophobic core of this HTH. Solvent-exposed amino acid residues located at many positions on the MarA surface are important for activity. Some of these residues affect activity on all promoters and thus, are implicated in maintaining MarA structure whereas several solvent-exposed amino acids not involved in DNA binding were important for MarA activity on specific promoters. The pattern of activation defects defined a class II promoter-specific activating region. However, a localized class I activating region was not apparent. These results suggest that MarA activates transcription by at least two distinct mechanisms. Furthermore, the important role of phosphate contacts in marbox affinity suggests that indirect readout contributes to binding site recognition by MarA.

• Moon MW et al.
• Cloning and expression of the ccpA gene encoding catabolite control protein from Thermoactinomyces sp. E79.
• Biosci Biotechnol Biochem. 2000; 64: 2254-8
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• The gene ccpA encoding the catabolite control protein A (CcpA) of Thermoactinomyces sp. E79 has been cloned and characterized. Nucleotide sequence analysis of the CcpA clone showed that the cloned fragment contained the full structural gene for a protein of 346 amino acids. The predicted amino acid sequence shows similarity to the transcriptional regulators of the Lacl-GalR family; a highly conserved helix-turn-helix motif, which might bind to DNA, was identified through comparison with regulator proteins in this family. The highest sequence identity was obtained when it was compared with the CcpA of Bacillus subtilis (60%) or Bacillus megaterium (60%). The expression of ccpA in Thermoactinomyces sp. E79 was dependent on glucose, which is contrast to the cases of B. subtilis, B. megaterium and S. xylosus. The complementation experiment with the B. megaterium ccpA mutant indicated that the cloned gene was a ccpA.

• Perez-Sanchez C, Arias-de-la-Fuente C, Gomez-Ferreria MA, Granadino B, Rey-Campos J
• FHX.L and FHX.S, two isoforms of the human fork-head factor FHX (FOXJ2) with differential activity.
• J Mol Biol. 2000; 301: 795-806
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• Many biological phenomena are dependent on mechanisms that fine-tune the expression levels of particular genes. This can be achieved by altering the relative activity of a single transcription factor, by post-translational modifications or by interaction with regulatory molecules. An alternative mechanism is based on competition between two or more differently active isoforms of the same transcription factor. We found that FHX, a recently characterized human fork-head transcriptional activator, may show such a mechanism for balancing its activity by expressing two differently sized isoforms, FHX.S and FHX.L, encoded by a single gene located on human chromosome 12. FHX. L and FHX.S showed different transcriptional capacities, the larger form, FHX.L, behaving as the more potent transactivator. A transactivation domain of the acidic type present only in FHX.L would account for this functional difference. The relative concentrations of these two FHX isoforms appear to vary in a number of cell types, a circumstance that may regulate the final activity of this transcription factor.

• Voo KS, Carlone DL, Jacobsen BM, Flodin A, Skalnik DG
• Cloning of a mammalian transcriptional activator that binds unmethylated CpG motifs and shares a CXXC domain with DNA methyltransferase, human trithorax, and methyl-CpG binding domain protein 1.
• Mol Cell Biol. 2000; 20: 2108-21
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• Ligand screening was utilized to isolate a human cDNA that encodes a novel CpG binding protein, human CpG binding protein (hCGBP). This factor contains three cysteine-rich domains, two of which exhibit homology to the plant homeodomain finger domain. A third cysteine-rich domain conforms to the CXXC motif identified in DNA methyltransferase, human trithorax, and methyl-CpG binding domain protein 1. A fragment of hCGBP that contains the CXXC domain binds to an oligonucleotide probe containing a single CpG site, and this complex is disrupted by distinct oligonucleotide competitors that also contain a CpG motif(s). However, hCGBP fails to bind oligonucleotides in which the CpG motif is either mutated or methylated, and it does not bind to single-stranded DNA or RNA probes. Furthermore, the introduction of a CpG dinucleotide into an unrelated oligonucleotide sequence is sufficient to produce a binding site for hCGBP. Native hCGBP is detected as an 88-kDa protein by Western analysis and is ubiquitously expressed. The DNA-binding activity of native hCGBP is apparent in electrophoretic mobility shift assays, and hCGBP trans-activates promoters that contain CpG motifs but not promoters in which the CpG is ablated. These data indicate that hCGBP is a transcriptional activator that recognizes unmethylated CpG dinucleotides, suggesting a role in modulating the expression of genes located within CpG islands.

• Manzanera M, Marques S, Ramos JL
• Mutational analysis of the highly conserved C-terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators.
• FEBS Lett. 2000; 476: 312-7
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• The XylS protein of the TOL plasmid of Pseudomonas putida belongs to the so-called AraC/XylS family of regulators, that includes more than 100 different bacterial proteins. A conserved stretch of about 100 amino acids is present at the C-terminal end. This conserved region is believed to contain seven alpha-helices, including two helix-turn-helix (HTH) DNA binding motifs (alpha(2)-T-alpha(3) and alpha(5)-Talpha-(6)), connected by a linker alpha-helix (alpha(4)), and two flanking alpha-helices (alpha(1) and alpha(7)). The second HTH motif is the region with the highest homology in the proteins of the family, with certain residues showing almost 90% identity. We have constructed XylS single mutants in the most conserved residues and have analysed their ability to stimulate transcription from its cognate promoter, Pm, fused to 'lacZ. The analysis revealed that mutations in the alpha(5)-helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the alpha(6)-helix was important for the activity. The strongest effect of the mutations was observed in conserved residues located outside the DNA binding domain, namely, Gly-290 in the turn between the two helices, Pro-309 located downstream of alpha(6), and Leu-313, in the small last helix alpha(7), that seems to play an important role in the activation of RNA-polymerase. Our analysis shows that conservation of amino acids in the family reflects structural requirements rather than functionality in specific DNA interactions.

• Watada H, Mirmira RG, Kalamaras J, German MS
• Intramolecular control of transcriptional activity by the NK2-specific domain in NK-2 homeodomain proteins.
• Proc Natl Acad Sci U S A. 2000; 97: 9443-8
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• The developmentally important homeodomain transcription factors of the NK-2 class contain a highly conserved region, the NK2-specific domain (NK2-SD). The function of this domain, however, remains unknown. The primary structure of the NK2-SD suggests that it might function as an accessory DNA-binding domain or as a protein-protein interaction interface. To assess the possibility that the NK2-SD may contribute to DNA-binding specificity, we used a PCR-based approach to identify a consensus DNA-binding sequences for Nkx2.2, an NK-2 family member involved in pancreas and central nervous system development. The consensus sequence (T(C)(T)AAGT(G)(A)(G)(C)TT) is similar to the known binding sequences for other NK-2 homeodomain proteins, but we show that the NK2-SD does not contribute significantly to specific DNA binding to this sequence. To determine whether the NK2-SD contributes to transactivation, we used GAL4-Nkx2. 2 fusion constructs to map a powerful transcriptional activation domain in the C-terminal region beyond the conserved NK2-SD. Interestingly, this C-terminal region functions as a transcriptional activator only in the absence of an intact NK2-SD. The NK2-SD also can mask transactivation from the paired homeodomain transcription factor Pax6, but it has no effect on transcription by itself. These results demonstrate that the NK2-SD functions as an intramolecular regulator of the C-terminal activation domain in Nkx2.2 and support a model in which interactions through the NK2-SD regulate the ability of NK-2-class proteins to activate specific genes during development.

• Soldaini E, John S, Moro S, Bollenbacher J, Schindler U, Leonard WJ
• DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites.
• Mol Cell Biol. 2000; 20: 389-401
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• We have defined the optimal binding sites for Stat5a and Stat5b homodimers and found that they share similar core TTC(T/C)N(G/A)GAA interferon gamma-activated sequence (GAS) motifs. Stat5a tetramers can bind to tandemly linked GAS motifs, but the binding site selection revealed that tetrameric binding also can be seen with a wide range of nonconsensus motifs, which in many cases did not allow Stat5a binding as a dimer. This indicates a greater degree of flexibility in the DNA sequences that allow binding of Stat5a tetramers than dimers. Indeed, in an oligonucleotide that could bind both dimers and tetramers, it was possible to design mutants that affected dimer binding without affecting tetramer binding. A spacing of 6 bp between the GAS sites was most frequently selected, demonstrating that this distance is favorable for Stat5a tetramer binding. These data provide insights into tetramer formation by Stat5a and indicate that the repertoire of potential binding sites for this transcription factor is broader than expected.

• Gillette WK, Rhee S, Rosner JL, Martin RG
• Structural homology between MarA of the AraC family of transcriptional activators and the integrase family of site-specific recombinases.
• Mol Microbiol. 2000; 35: 1582-3

• Steinmetzer K, Hillisch A, Behlke J, Brantl S
• Transcriptional repressor CopR: structure model-based localization of the deoxyribonucleic acid binding motif.
• Proteins. 2000; 38: 393-406
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• The plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'-CGTG. Intermediate sequence searches revealed a significant structural relationship between CopR and the bacteriophage P22 c2 and the 434 c1 repressors. In this report we describe the experimental verification of a CopR homology model, which is based on a fairly low-sequence identity of 13.8% to P22 c2 repressor. A model for the complex of CopR with the deoxyribonucleic acid (DNA) target was built on the basis of experimental footprinting data, the above-mentioned CopR homology model, and the crystal structure of the 434 c1 repressor-DNA complex. Site-directed mutagenesis was used to test the function of amino acids involved in sequence and nonsequence-specific DNA recognition and amino acids important for correct protein folding. CD measurements were performed to detect structural changes caused by the mutations. Exchanges of residues responsible for sequence-specific DNA recognition reduced binding to a nonspecific level. Mutations of amino acids involved in nonspecific DNA binding lead to decreased binding affinity while maintaining selectivity. Substitution of amino acids necessary for proper folding caused dramatic structural changes. The experimental data support the model of CopR as a helix-turn-helix protein belonging to the lambda repressor superfamily.

• Kaldalu N, Toots U, de Lorenzo V, Ustav M
• Functional domains of the TOL plasmid transcription factor XylS.
• J Bacteriol. 2000; 182: 1118-26
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• The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

• Eichelberg K, Hardt WD, Galan JE
• Characterization of SprA, an AraC-like transcriptional regulator encoded within the Salmonella typhimurium pathogenicity island 1.
• Mol Microbiol. 1999; 33: 139-52
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• Pathogenicity island 1 (SPI-1) located at centisome 63 of the Salmonella chromosome encodes a type III protein secretion system that is essential for its pathogenicity. The translocation of effector proteins through this system results in the stimulation of signalling events, leading to actin cytoskeletal rearrangements and nuclear responses. These cellular responses ultimately lead to bacterial uptake, production of proinflammatory cytokines in non-phagocytic cells and the initiation of programmed cell death in macrophages. The regulation of expression of components and substrates of this type III secretion system is complex and involves the activity of several specific transcriptional regulatory proteins encoded within SPI-1. Here, we describe two additional regulatory proteins, SprA and SprB, which are encoded within SPI-1. SprA and SprB exhibit significant sequence similarity to the AraC/XylS and the LuxR/UhaP family of transcriptional regulatory proteins respectively. Insertion mutations in sprA and sprB did not significantly affect the transcription of invasion-associated genes and, consequently, did not affect the ability of Salmonella typhimurium to gain access into host cells. However, expression of sprA from an inducible heterologous promoter resulted in increased expression of genes associated with the centisome 63 type III secretion system and increased the ability of S. typhimurium to enter into host cells. Further analysis demonstrated that SprA acts either upstream or at the same level as HilA in the SPI-1 transcriptional regulatory cascade.

• Christian JL, Nakayama T
• Can't get no SMADisfaction: Smad proteins as positive and negative regulators of TGF-beta family signals.
• Bioessays. 1999; 21: 382-90
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• The identification of Smad proteins as molecular components of the transforming growth factor-beta (TGF-beta) signaling cascade has enhanced our understanding of how ligand-mediated activation of TGF-beta receptors leads to modulation of target gene transcription. Recent studies have identified a distinct, structurally related class of Smads which inhibits, rather than transduces, TGF-beta family signals. The molecular mechanism of action and the exact signaling pathways that are targeted by antagonistic Smads are not completely understood. These proteins appear to participate in autoregulatory negative feedback loops in which signaling initiated by specific TGF-beta family ligands induces the expression of an inhibitory Smad that then functions to modulate the amplitude or duration of signaling. Negative feedback circuits such as these play important roles in fine-tuning the activity of multifunctional signaling molecules during embryonic patterning and in response to pathologic stimuli in adults.

• Cramer P, Varrot A, Barillas-Mury C, Kafatos FC, Muller CW
• Structure of the specificity domain of the Dorsal homologue Gambif1 bound to DNA.
• Structure Fold Des. 1999; 7: 841-52
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• BACKGROUND: NF-kappa B/Rel transcription factors play important roles in immunity and development in mammals and insects. Their activity is regulated by their cellular localization, homo- and heterodimerization and association with other factors on their target gene promoters. Gambif1 from Anopheles gambiae is a member of the Rel family and a close homologue of the morphogen Dorsal, which establishes dorsoventral polarity in the Drosophila embryo. RESULTS: We present the crystal structure of the N-terminal specificity domain of Gambif1 bound to DNA. This first structure of an insect Rel protein-DNA complex shows that Gambif1 binds a GGG half-site element using a stack of three arginine sidechains. Differences in affinity to Dorsal binding sites in target gene promoters are predicted to arise from base changes in these GGG elements. An arginine that is conserved in class II Rel proteins (members of which contain a transcription activation domain) contacts the outermost guanines of the DNA site. This previously unseen specific contact contributes strongly to the DNA-binding affinity and might be responsible for differences in specificity between Rel proteins of class I and II. CONCLUSIONS: The Gambif1-DNA complex structure illustrates how differences in Dorsal affinity to binding sites in developmental gene promoters are achieved. Comparison with other Rel-DNA complex structures leads to a general model for DNA recognition by Rel proteins.

• Ozaki J et al.
• Identification of the core domain and the secondary structure of the transcriptional coactivator MBF1.
• Genes Cells. 1999; 4: 415-24
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• BACKGROUND: Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ-F1 and GCN4. MBF1 bridges the DNA-binding regions of these activators and the TATA-box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP. RESULTS: The two-dimensional (2D) 1H-15N correlation spectrum of 15N labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts. Limited digestion of MBF1 by alpha-chymotrypsin yielded a approximately 9 kDa fragment. N-terminal sequence analysis and NMR measurements revealed that this fragment originates from the C-terminal 80 residues of MBF1 and form a well structured C-terminal domain of MBF1, MBF1CTD. As previous deletion analyses have shown that MBF1CTD is capable of binding to TBP, it is suggested that MBF1CTD is the TBP binding domain of MBF1. Sequential assignments have been obtained by means of three-dimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1CTD was determined. As a result, MBF1CTD was shown to contain four amphipathic helices and a conserved C-terminal region. Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix. CONCLUSIONS: Structural analyses revealed that MBF1 consists of two structurally different domains. A N-terminal region is indispensable for the binding to activators, and does not form a well defined structure. In contrast, the C-terminal 80 residues, which is capable of binding to TBP by itself, form a well-structured domain, MBF1CTD. MBF1CTD is made up of four amphipathic helices and a conserved C-terminal tail. A putative TBP binding residue is located on the hydrophilic surface of the third helix.

• Eroshkin A, Mushegian A
• Conserved transactivation domain shared by interferon regulatory factors and Smad morphogens.
• J Mol Med. 1999; 77: 403-5
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• Interferon regulatory factors (IRFs) regulate the transcription of both interferon-inducible genes and interferons themselves. Along with the N-terminal, DNA-binding, winged-helix domain, most IRFs contain the C-terminal domains that are shown to be related to the C-terminal domains in the proteins of the Smad family that mediate transcription activation in the transforming growth factor response pathway. Comparison of the IRF-Smad alignment to the known three-dimensional structure of human tumor suppressor Smad4 suggests that a conserved loop, equivalent to Loop 3 in Smad 4, is a determinant of protein-protein interaction in IRFs.

• Bhende PM, Egan SM
• Amino acid-DNA contacts by RhaS: an AraC family transcription activator.
• J Bacteriol. 1999; 181: 5185-92
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• RhaS, an AraC family protein, activates rhaBAD transcription by binding to rhaI, a site consisting of two 17-bp inverted repeat half-sites. In this work, amino acids in RhaS that make base-specific contacts with rhaI were identified. Sequence similarity with AraC suggested that the first contacting motif of RhaS was a helix-turn-helix. Assays of rhaB-lacZ activation by alanine mutants within this potential motif indicated that residues 201, 202, 205, and 206 might contact rhaI. The second motif was identified based on the hypothesis that a region of especially high amino acid similarity between RhaS and RhaR (another AraC family member) might contact the nearly identical DNA sequences in one major groove of their half-sites. We first made targeted, random mutations and then made alanine substitutions within this region of RhaS. Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding. A genetic loss-of-contact approach was used to identify whether any of the RhaS amino acids in the first or second contacting motif make base-specific DNA contacts. In motif 1, we found that Arg202 and Arg206 both make specific contacts with bp -65 and -67 in rhaI1, and that Arg202 contacts -46 and Arg206 contacts -48 in rhaI2. In motif 2, we found that Asp250 and Asn252 both contact the bp -79 in rhaI1. Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.

• Ohnishi Y, Kameyama S, Onaka H, Horinouchi S
• The A-factor regulatory cascade leading to streptomycin biosynthesis in Streptomyces griseus : identification of a target gene of the A-factor receptor.
• Mol Microbiol. 1999; 34: 102-11
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• In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) at an extremely low concentration triggers streptomycin biosynthesis and cell differentiation by binding a repressor-type receptor protein (ArpA) and dissociating it from DNA. An A-factor-responsive transcriptional activator (AdpA) able to bind the promoter of strR, a pathway-specific regulatory gene responsible for transcription of other streptomycin biosynthetic genes, was purified to homogeneity and adpA was cloned by PCR on the basis of amino acid sequences of purified AdpA. adpA encoding a 405-amino-acid protein containing a helix-turn-helix DNA-binding motif at the central region showed sequence similarity to transcriptional regulators in the AraC/XylS family. The -35 and -10 regions of the adpA promoter were found to be a target of ArpA; ArpA bound the promoter region in the absence of A-factor and exogenous addition of A-factor to the DNA-ArpA complex immediately released ArpA from the DNA. Consistent with this, S1 nuclease mapping showed that adpA was transcribed only in the presence of A-factor and strR was transcribed only in the presence of intact adpA. Furthermore, adpA disruptants produced no streptomycin and overexpression of adpA caused the wild-type S. griseus strain to produce streptomycin at an earlier growth stage in a larger amount. On the basis of these findings, we propose here a model to demonstrate how A-factor triggers streptomycin biosynthesis at a late exponential growth stage.

• Xie Q, Sanz-Burgos AP, Guo H, Garcia JA, Gutierrez C
• GRAB proteins, novel members of the NAC domain family, isolated by their interaction with a geminivirus protein.
• Plant Mol Biol. 1999; 39: 647-56
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• Geminiviruses encode a few proteins and depend on cellular factors to complete their replicative cycle. As a way to understand geminivirus-host interactions, we have searched for cellular proteins which interact with viral proteins. By using the yeast two-hybrid technology and the wheat dwarf geminivirus (WDV) RepA protein as a bait, we have isolated a family of proteins which we termed GRAB (for Geminivirus Rep A-binding). We report here the molecular characterization of two members, GRAB1 and GRAB2. We have found that the 37 C-terminal amino acids of RepA are required for interaction with GRAB proteins. This region contains residues conserved in an equivalent region of the RepA proteins encoded by other viruses of the WDV subgroup. The N-terminal domain of GRAB proteins is necessary and sufficient to interact with WDV RepA. GRAB proteins contain an unique acidic C-terminal domain while their N-terminal domain, of ca. 170 amino acids, are highly conserved in all of them. Interestingly, this conserved N-terminal domain of GRAB proteins exhibits a significant amino acid homology to the NAC domain present in proteins involved in plant development and senescence. GRAB1 and GRAB2 mRNAs are present in cultured cells and roots but are barely detectable in leaves. GRAB expression inhibits WDV DNA replication in cultured wheat cells. Our studies highlight the importance that the pathway(s) mediated by GRAB proteins, as well as by other NAC domain-containing proteins, might have on geminivirus DNA replication in connection to plant growth, development and senescence pathways.

• Reed WL, Schleif RF
• Hemiplegic mutations in AraC protein.
• J Mol Biol. 1999; 294: 417-25
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• We have isolated mutations in AraC protein that specifically block either induction or repression at the ara pBAD promoter. These hemiplegic mutations identify amino acid residues that, correspondingly, are involved only in the induction or only in the repression activities of the protein. Residues key only for induction are 13, 15, and 18, which are located in the N-terminal arm of AraC, and residues 80 and 82 which lie in the arabinose-binding pocket of the protein's sugar-binding and dimerization domain. Alteration of residues 157, 244 and 257 can leave the protein able to activate transcription but not able to repress transcription. The behavior of the mutant proteins is consistent with the light switch mechanism for AraC action in which the presence of arabinose pulls the N-terminal arms of the protein off the DNA-binding domains, thereby freeing them to assume a direct-repeat orientation, bind to adjacent direct-repeat DNA half-sites, and activate transcription.

• Decker T, Kovarik P
• Transcription factor activity of STAT proteins: structural requirements and regulation by phosphorylation and interacting proteins.
• Cell Mol Life Sci. 1999; 55: 1535-46
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• The seven mammalian members of the signal transducer and activator of transcription (STAT) family share a common core structure which reflects their shared mechanism of activation, dimerization, and DNA binding. By contrast, the STAT C termini containing the sequences required for transcriptional activation are much less homologous, suggesting different ways by which individual STATs activate their target genes. This paper describes several important discoveries linked to mechanistic aspects of STAT transcription factor function. These include regulated serine phosphorylation of the transactivating domain, promoter-dependent interactions of STATs with each other, or of STATs with other transcription factors, and with transcriptional co-activators. The basis, background, and implications of these molecular events will be summarized and discussed.

• Schreiber J, Enderich J, Wegner M
• Structural requirements for DNA binding of GCM proteins.
• Nucleic Acids Res. 1998; 26: 2337-43
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• Members of the GCM family of transcription factors contain a DNA binding domain unrelated to any other known DNA binding domain and bind to a DNA sequence motif not recognized by any other known transcription factor. Here we show that positions 2, 3, 6 and 7 of the 5'-ATGCGGGT-3' motif are particularly important for DNA binding and that methylation of several G residues on the upper strand, but not on the lower strand, interfered with binding of GCM proteins. No differences were detected between the DNA binding of Drosophila GCM and mammalian mGCMa. Alanine scan mutagenesis of the DNA binding domain of mGCMa identified the three conserved amino acids K74, C76 and C125 as being essential for DNA binding. Conserved cysteine residues were also found to be important for maintaining the overall integrity of the DNA binding domain and for mediating redox sensitivity of DNA binding. These cysteine residues are arranged in a symmetrical structure that bears no resemblance to other cysteine-containing structures, such as zinc fingers. In agreement with this, DNA binding of mGCMa was not dependent on zinc ions. Our results give insights into the exact nature of the GCM binding sites expected in target genes and point to a role for redox regulation in the function of GCM proteins.

• Schroder P, Meyer L, Wheeler TT, Thiesen HJ, Seyfert HM
• Cloning and sequencing of the bovine STAT5A cDNA reveals significant sequence divergence with ovine.
• Biochim Biophys Acta. 1998; 1398: 99-105
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• The transcription factors STAT5 mediates prolactin signals in mammary epithelial cells. The cDNA of bovine STAT5A was cloned, sequenced and compared to other species. The encoded protein proves to be > 95% homologous to other mammals. We show that the STAT5A mRNA of the closely related ovine species contains an extended (by 130 nt) 5'-untranslated region, being encoded by an extra-exon, and accounts, possibly, for improved translation efficiency.

• Turner J, Crossley M
• Cloning and characterization of mCtBP2, a co-repressor that associates with basic Kruppel-like factor and other mammalian transcriptional regulators.
• EMBO J. 1998; 17: 5129-40
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• Basic Kruppel-like factor (BKLF) is a zinc finger protein that recognizes CACCC elements in DNA. It is expressed highly in erythroid tissues, the brain and other selected cell types. We have studied the activity of BKLF and found that it is capable of repressing transcription, and have mapped its repression domain to the N-terminus. We carried out a two-hybrid screen against BKLF and isolated a novel clone encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein which binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral oncoprotein, E1a. We show that mCtBP2 recognizes a related motif in the minimal repression domain of BKLF, and the integrity of this motif is required for repression activity. Moreover, when tethered to a promoter by a heterologous DNA-binding domain, mCtBP2 functions as a potent repressor. Finally, we demonstrate that mCtBP2 also interacts with the mammalian transcripition factors Evi-1, AREB6, ZEB and FOG. These results establish a new member of the CtBP family, mCtBP2, as a mammalian co-repressor targeting diverse transcriptional regulators.

• Lam LT, Bresnick EH
• Identity of the beta-globin locus control region binding protein HS2NF5 as the mammalian homolog of the notch-regulated transcription factor suppressor of hairless.
• J Biol Chem. 1998; 273: 24223-31
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• Previously, we characterized a DNA-binding protein, HS2NF5, that bound tightly to a conserved region within hypersensitive site 2 (HS2) of the human beta-globin locus control region (LCR) (Lam, L. T. , and Bresnick, E. H. (1996) J. Biol. Chem. 271, 32421-32429). The beta-globin LCR controls the chromatin structure, transcription, and replication of the beta-globin genes. We have now purified HS2NF5 to near-homogeneity from fetal bovine thymus. Two polypeptides of 56 and 61 kDa copurified with the DNA binding activity. The two proteins bound to the LCR recognition site with an affinity (3.1 nM) and specificity similar to mouse erythroleukemia cell HS2NF5. The amino acid sequences of tryptic peptides of purified HS2NF5 revealed it to be identical to the murine homolog of the suppressor of hairless transcription factor, also known as recombination signal binding protein Jkappa or C promoter binding factor 1 (CBF1). The CBF1 site within HS2 resides near sites for hematopoietic regulators such as GATA-1, NF-E2, and TAL1. An additional conserved, high affinity CBF1 site was localized within HS4 of the LCR. As CBF1 is a downstream target of the Notch signaling pathway, we propose that Notch may modulate LCR activity during hematopoiesis.

• Kherrouche Z et al.
• Isolation and characterization of a chicken homologue of the Spi-1/PU.1 transcription factor.
• Oncogene. 1998; 16: 1357-67
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• Spi-1/PU.1 is a member of the Ets family of transcription factors important in regulation of hematopoiesis. We have isolated a chicken cDNA homologuous to the mammalian Spi-1/PU.1 gene with an open reading frame of 250 amino acids (aa). The chicken Spi-1/PU.1 protein is 14 aa and 16 aa shorter than its human and mouse counterparts but is extremely well conserved with 78.8% and 75.2% identity respectively. The carboxy terminal DNA binding region, or ETS binding domain, is 100% identical to that of human and mouse. Some differences with the mammalian homologues are seen in the N-terminal part of the protein and in the PEST connecting domain. However, the differences are mainly conservative and all the features underlying functional aspects seem preserved. The major discrepancy lies in a 12 aa deletion in an already poorly conserved part of the PEST sequence. Spi-1/PU.1 transcripts were detected at high levels in spleen and Fabricius bursa of chick embryos by Northern blot and in situ hybridization. Our results show that the chicken Spi-1/PU.1 protein behaves like a bonafide Spi-1/PU.1 transcription factor in its DNA binding and transactivating properties.

• Hoe KL et al.
• Molecular cloning of gaf1, a Schizosaccharomyces pombe GATA factor, which can function as a transcriptional activator.
• Gene. 1998; 215: 319-28
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• As a first step to elucidate the functions of Schizosaccharomyces pombe (S. pombe) GATA factors, we have isolated the gaf1+ gene (GATA-factor like gene) in S. pombe. The predicted amino acid (aa) sequence of Gaf1 reveals a single zinc finger domain typical of fungal GATA factors, and the zinc finger exhibits 60% aa identity to that of human GATA-1. The open reading frame of Gaf1 predicts a protein of Mr 32 kDa consisting of 290 intronless amino acids. Disruption of this gene has no effect on cell viability and growth rate. The GST-Gaf1 fusion protein binds specifically to GATA motifs of its own promoter as well as DAL7 UAS, a canonical GATA motif of Saccharomyces cerevisiae (S. cerevisiae) The specific DNA-binding activity resides within the N-terminal half of Gaf1 (Gaf1N; aa 1-120) containing the zinc finger, whereas the C-terminal half (Gaf1C; aa 121-290) contains transactivation sequences that induce the expression of the lacZ reporter when fused to the GAL4 DNA binding domain. These results demonstrate that Gaf1 may function as a transcriptional activator consisting of DNA-binding and transactivation domains.

• Liu QX, Ueda H, Hirose S
• Comparison of sequences of a transcriptional coactivator MBF2 from three Lepidopteran species Bombyx mori, Bombyx mandarina and Samia cynthia.
• Gene. 1998; 220: 55-9
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• MBF2 was first isolated from the silkworm Bombyx mori as a positive cofactor that activates transcription through its interaction with TFIIA. To identify conserved domain(s) within the MBF2 molecule, we isolated cDNAs encoding MBF2 homologues from other silkworms Bombyx mandarina and Samia cynthia. Bacterially expressed and purified MBF2 of B. mandarina and S. cynthia activated transcription in vitro. The predicted amino acid sequences of MBF2 from two Bombyx species share 97% homology. When we compared between B. mori and S. cynthia factors, the homology reduced to 50%. Four regions in MBF2 are conserved among these three species. Two of them are present in the middle region of MBF2 that is essential for the transcriptional activation.

• Pietrokovski S, Henikoff S
• A helix-turn-helix DNA-binding motif predicted for transposases of DNA transposons.
• Mol Gen Genet. 1997; 254: 689-95
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• A helix-turn-helix (HTH) DNA-binding motif is identified in transposase sequences in Tc1, mariner and pogo DNA transposum. The findings are supported by results of various sequence analysis methods. Tc1 transposases are also predicted to contain another DNA-binding region. These findings are in accord with experimental evidence obtained from Tc1A, Tc3A and pogo transposases. The pogo family transposases, but not the pogo-type transcription factors, contain the HTH motif, suggesting that HTH structures are essential for Tc1/mariner/pogo transposition. Analysis of multiple sequence alignments enabled the identification of the HTH motif in distantly related protein sequences.

• Martinez-Hackert E, Stock AM
• The DNA-binding domain of OmpR: crystal structures of a winged helix transcription factor.
• Structure. 1997; 5: 109-24
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• BACKGROUND: The differential expression of the ompF and ompC genes is regulated by two proteins that belong to the two component family of signal transduction proteins: the histidine kinase, EnvZ, and the response regulator, OmpR. OmpR belongs to a subfamily of at least 50 response regulators with homologous C-terminal DNA-binding domains of approximately 98 amino acids. Sequence homology with DNA-binding proteins of known structure cannot be detected, and the lack of structural information has prevented understanding of many of this familys functional properties. RESULTS: We have determined the crystal structure of the Escherichia coli OmpR C-terminal domain at 1.95 A resolution. The structure consists of three alpha helices packed against two antiparallel beta sheets. Two helices, alpha2 and alpha3, and the ten residue loop connecting them constitute a variation of the helix-turn-helix (HTH) motif. Helix alpha3 and the loop connecting the two C-terminal beta strands, beta6 and beta7, are probable DNA-recognition sites. Previous mutagenesis studies indicate that the large loop connecting helices alpha2 and alpha3 is the site of interaction with the alpha subunit of RNA polymerase. CONCLUSIONS: OmpRc belongs to the family of 'winged helix-turn-helix' DNA-binding proteins. This relationship, and the results from numerous published mutagenesis studies, have helped us to interpret the functions of most of the structural elements present in this protein domain. The structure of OmpRc could be useful in helping to define the positioning of the alpha subunit of RNA polymerase in relation to transcriptional activators that are bound to DNA.

• Wako H, Tachikawa M, Ogawa A
• A comparative study of dynamic structures between phage 434 Cro and repressor proteins by normal mode analysis.
• Proteins. 1996; 26: 72-80
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• Two DNA binding proteins, Cro and the amino-terminal domain of the repressor of bacteriophage 434 (434 Cro and 434 repressor) that regulate gene expression and contain a helix-turn-helix (HTH) motif responsible for their site-specific DNA recognition adopt very similar three-dimensional structures when compared to each other. To reveal structural differences between these two similar proteins, their dynamic structures, as examined by normal mode analysis, are compared in this paper. Two kinds of structural data, one for the monomer and the other for a complex with DNA, for each protein, are used in the analyses. From a comparison between the monomers it is found that the interactions of Ala-24 in 434 Cro or Val-24 in 434 repressor, both located in the HTH motif, with residues 44, 47, 48, and 51 located in the domain facing the motif, and the interactions between residues 17, 18, 28, and 32, located in the HTH motif, cause significant differences in the correlative motions of these residues. From the comparison between the monomer and the complex with DNA for each protein, it was found that the first helix in the HTH motif is distorted in the complex form. While the residues in the HTH motif in 434 Cro have relatively larger positive correlation coefficients of motions with other residues within the HTH motif, such correlations are not large in the HTH motif of 434 repressor. It is suggestive to their specificity because the 434 repressor is less specific than 434 Cro. Although a structural comparison of proteins has been performed mainly from a static or geometrical point of view, this study demonstrates that the comparison from a dynamic point of view, using the normal mode analysis, is useful and convenient to explore a difference that is difficult to find only from a geometrical point of view, especially for proteins very similar in structure.

• Hachler H, Cohen SP, Levy SB
• Untranslated sequence upstream of MarA in the multiple antibiotic resistance locus of Escherichia coli is related to the effector-binding domain of the XylS transcriptional activator.
• J Mol Evol. 1996; 42: 409-13
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• MarA, the 129-amino-acid (aa) protein which plays a crucial role in the multiple antibiotic resistance (Mar) phenotype in Escherichia coli, shows homology to members of the XylS/AraC family of transcriptional regulators. Although these proteins vary in size from around 100 to 350 aa they all contain a DNA-binding domain with a helix-turn-helix motif. The larger ones, e.g., XylS, AraC, and Rob, contain an additional domain either at their amino- or at their carboxyterminus. This domain is important for effector-binding or dimerization or of unknown function. MarA consists only of the DNA-binding component. Nevertheless, a sequence with a coding potential of 141 aa upstream of its ATG start-codon showed 20.5-26.9% aa identity with the corresponding section within the effector-binding domain of XylS from the TOL plasmid of Pseudomonas putida when translated in the same reading frame as MarA. However, the reading frame was interrupted by 11 translational stops. In another frame, this upstream sequence actually codes for a real protein, MarR, that is completely unrelated to XylS. Implications for the putative evolution of regulatory proteins through translocation of domains followed by adaptation are discussed.

• Zeng G, Ye S, Larson TJ
• Repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K-12: primary structure and identification of the DNA-binding domain.
• J Bacteriol. 1996; 178: 7080-9
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• The nucleotide sequence of the glpEGR operon of Escherichia coli was determined. The translational reading frame at the beginning, middle, and end of each gene was verified. The glpE gene encodes an acidic, cytoplasmic protein of 108 amino acids with a molecular weight of 12,082. The glpG gene encodes a basic, cytoplasmic membrane-associated protein of 276 amino acids with a molecular weight of 31,278. The functions of GlpE and GlpG are unknown. The glpR gene encodes the repressor for the glycerol 3-phosphate regulon, a protein predicted to contain 252 amino acids with a calculated molecular weight of 28,048. The amino acid sequence of the glp repressor was similar to several repressors of carbohydrate catabolic systems, including those of the glucitol (GutR), fucose (FucR), and deoxyribonucleoside (DeoR) systems of E. coli, as well as those of the lactose (LacR) and inositol (IolR) systems of gram-positive bacteria and agrocinopine (AccR) system of Agrobacterium tumefaciens. These repressors constitute a family of related proteins, all of which contain approximately 250 amino acids, possess a helix-turn-helix DNA-binding motif near the amino terminus, and bind a sugar phosphate molecule as the inducing signal. The DNA recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system. The presumptive recognition helix of the glp repressor was changed by site-directed mutagenesis to match that of the deo repressor or, in a separate construct, to abolish DNA binding. Neither altered form of the glp repressor recognized the glp or deo operator, either in vivo or in vitro. However, both altered forms of the glp repressor were negatively dominant to the wild-type glp repressor, indicating that the inability to bind DNA with high affinity was due to alteration of the DNA-binding domain, not to an inability to oligomerize or instability of the altered repressors. For the first time, analysis of repressors with altered DNA-binding domains has verified the assignment of the helix-turn-helix motif of the transcriptional regulators in the deoR family.

• de Pater S, Greco V, Pham K, Memelink J, Kijne J
• Characterization of a zinc-dependent transcriptional activator from Arabidopsis.
• Nucleic Acids Res. 1996; 24: 4624-31
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• The C2-H2 zinc-finger is a widely occurring DNA binding motif, usually present as tandem repeats. The majority of C2-H2 zinc-finger proteins that have been studied are derived from animals. Here, we characterize a member of a distinct class of plant C2-H2 zinc-finger proteins in detail. A cDNA clone encoding a DNA binding protein from Arabidopsis was isolated by SouthWestern screening. The protein, termed ZAP1 (Zinc-dependent Activator Protein-1), is encoded by a single copy gene, which is expressed to similar levels in root and flower, to a somewhat lower level in stem and to low levels in leaf and siliques. The optimal binding site was determined by random binding site selection, and the consensus sequence found is CGTTGACCGAG. The homology between ZAP1 and other DNA binding proteins is restricted to a repeated region of a stretch of 24 highly conserved amino acids followed by a zinc-finger motif (C-X4-C-X22-23-H-X1-H). The C-terminal zinc-finger region is essential for DNA binding, whereas deletion of the N-terminal one resulted in 2.5-fold reduced binding affinity. Binding of ZAP1 to DNA was abolished by metal-chelating agents. The activation domain as determined in yeast is adjacent to and possibly overlapping with the DNA binding domain. Particle bombardment experiments with plant cells showed that ZAP1 increases expression of a gusA reporter gene that is under control of ZAP1 binding sites. We conclude that ZAP1 is a plant transcriptional activator with a C2-H2 zinc-finger DNA binding domain.

• Sheehan B, Klarsfeld A, Ebright R, Cossart P
• A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence.
• Mol Microbiol. 1996; 20: 785-97
• Display abstract

• PrfA, the regulator of virulence-gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP-FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of PrfA predicted to contact DNA, i.e. Ser-184 and Ser-183. Substitution of Ser-184 by Ala reduced DNA binding and virulence-gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser-183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target-site recognition by PrfA and PrfA-S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence-gene activation, and virulence.

• Holm L et al.
• LexA repressor and iron uptake regulator from Escherichia coli: new members of the CAP-like DNA binding domain superfamily.
• Protein Eng. 1994; 7: 1449-53
• Display abstract

• Comparison of structures can reveal surprising connections between protein families and provide new insights into the relationship between sequence, structure and function. The solution structure of LexA repressor from Escherichia coli reveals an unexpected structural similarity to a widespread class of prokaryotic and eukaryotic regulatory proteins, which is typified by catabolite gene activator protein (CAP). The use of combined sequence profiles allows the identification of two new prokaryotic members of the superfamily: listeriolysin regulatory protein (PrfA) and ferric uptake regulatory protein (Fur). LexA, PrfA and Fur are the first examples of prokaryotic regulatory proteins in which DNA recognition is mediated by a variant of the classical helix-turn-helix motif, with an insertion in the turn region.

• Schumacher MA, Macdonald JR, Bjorkman J, Mowbray SL, Brennan RG
• Structural analysis of the purine repressor, an Escherichia coli DNA-binding protein.
• J Biol Chem. 1993; 268: 12282-8
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• The purine repressor protein, PurR, is a member of the lac repressor, LacI, family of Escherichia coli DNA-binding proteins that bind DNA via a highly conserved N-terminal helix-turn-helix motif. Additionally, the members of this family display strong sequence homologies between their larger C-terminal effector binding/oligomerization domains. Analysis of the PurR primary and secondary structures reveals that its C-terminal corepressor binding domain is highly homologous to another group of E. coli-binding proteins, the periplasmic binding proteins, particularly to the ribose-binding protein (RBP). The high resolution x-ray structure of RBP allows this protein to serve as a template with which to model the predicted secondary structure of the corepressor binding domain of PurR. Similarly, the N-terminal DNA binding domain of PurR can be modeled using the NMR-determined structure of the corresponding region (residues 1-56) from LacI as a template. Combining the two, results in a description of the likely secondary structure topology of PurR and implicates residues important for corepressor binding and dimerization. CD spectroscopic studies on PurR, its corepressor binding domain and RBP result in secondary structure estimates nearly identical with those obtained by sequence analyses, thereby providing further corroborating physical evidence for this topological assignment.

• Bairoch A
• A possible mechanism for metal-ion induced DNA-protein dissociation in a family of prokaryotic transcriptional regulators.
• Nucleic Acids Res. 1993; 21: 2515-2515

• Huckle JW, Morby AP, Turner JS, Robinson NJ
• Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions.
• Mol Microbiol. 1993; 7: 177-87
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• In eukaryotes, metallothioneins (MTs) are involved in cellular responses to elevated concentrations of certain metal ions. We report the isolation and analysis of a prokaryotic MT locus from Synechococcus PCC 7942. The MT locus (smt) includes smtA, which encodes a class II MT, and a divergently transcribed gene, smtB. The sites of transcription initiation of both genes have been mapped and features within the smt operator-promoter region identified. Elevated concentrations of the ionic species of Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn elicited an increase in the abundance of smtA transcripts. There was no detectable effect of elevated metal (Cd) on smtA transcript stability. Sequences upstream of smtA, fused to a promoterless lacZ gene, conferred metal-dependent beta-galactosidase activity in Synechococcus PCC 7942 (strain R2-PIM8). At maximum permissive concentrations, Zn was the most potent elicitor in vivo, followed by Cu and Cd with slight induction by Co and Ni. The deduced SmtB polypeptide has similarity to the ArsR and CadC proteins involved in resistance to arsenate/arsenite/antimonite and to Cd, contains a predicted helix-turn-helix DNA-binding motif and is shown to be a repressor of transcription from the smtA operator-promoter.

• Ramos JL, Rojo F, Zhou L, Timmis KN
• A family of positive regulators related to the Pseudomonas putida TOL plasmid XylS and the Escherichia coli AraC activators.
• Nucleic Acids Res. 1990; 18: 2149-52
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• The XylS family consists of a least 8 different transcriptional regulators. Six of these proteins are positive regulators for the catabolism of carbon sources (benzoate and sugars) in Escherichia coli, Pseudomonas putida and Erwinia carotovora, and two of them are involved in pathogenesis in Escherichia coli and Yersinia enterocolitica. Based on protein alignments, the members of this family exhibit a long stretch of homology at the C-terminal end. The regulators involved in the catabolism of carbon sources stimulate transcription from their respectively regulated promoters only in the presence of effectors. In two of the regulators, mutations at the non-homologous N-terminus alter affinity and specificity for effectors while mutations at the conserved C-terminus part decrease activation of transcription from their corresponding regulated promoters. It is thus probable that the variable N-terminus end in this family of regulators contains the motif involved in effector recognition, while the C-terminal end is involved in DNA-binding. These proteins seem to be related by common ancestry and may act through similar mechanisms of positive regulation effected through similar folding patterns.

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