The DNA-binding domain of the c-myb protooncogene product consists of three imperfect tandem repeats of 51 or 52 amino acids, each of which contains three conserved tryptophans, spaced 18 or 19 amino acids apart. The structure ofthe third repeat, which is essential for sequence-specific DNA binding, has been determined by NMR with distance geometry calculation. It includes three well-defined helices (residues 149-162, 166-172, and 178-187) maintained by a hydrophobic core that includes the three conserved tryptophans, together with two histidines. Helices 2 and 3 form a structure related to but distinct from a canonical helix-turn-helix motif. In particular, the turn between these helices is one amino acid longer than the corresponding turn in bacterial repressors and homeodomains and contains a proline residue. In addition, the architecture of the three helices is different from those of homeodomains and DNA-binding domains of bacterial repressors. Based on the present structure, the binding mode of Myb repeat 3 with a specific DNA is also discussed.The c-myb protooncogene product (c-Myb) binds to DNA in a sequence-specific manner and acts as a transcriptional regulator (1)(2)(3)(4)(5). The DNA-binding domain of c-Myb consists of three imperfect tandem repeats of 51 or 52 amino acids (6-8), which are reported to have a similarity with the homeodomain (9). It has been suggested that the third repeat recognizes the specific base sequence, whereas the first repeat cooperates to bind to DNA (8,(10)(11)(12). The role of the second repeat is unclear. It was suggested that the second repeat could also participate in recognition of a specific base sequence by assuming that the second and third repeats take a similar structure related to the helix-turn-helix (HTH) motif (12). However, no three-dimensional structures of these repeats have been established. In addition, each repeat contains three conserved tryptophans, spaced 18 or 19 amino acids apart, which play a critical role in sequence-specific DNA binding (10,11,13). A conserved triplet of tryptophans with spacing similar to that of c-Myb is also found in the DNA-binding domains of the products of ets gene family. This indicates that these conserved tryptophans may represent a characteristic property of a group of DNA-binding proteins. To clarify the role of three tryptophans as well as to find the exact role of three repeats, we have determined the three-dimensional structure of the third repeat in aqueous solution by NMR. MATERIALS AND METHODSSample Preparation. A peptide corresponding to the third repeat of the DNA-binding domain of mouse c-Myb, amino acids 142-193, was chemically synthesized and purified by reversed-phase high-performance liquid chromatography as described (14). The purity of the synthesized c-Myb-(142-193)-NH2 was examined by ion-exchange chromatography as well as by amino acid composition analysis; no contamination by side products was detected (14).NMR Measurements. The lyophilized sample was dissolved in a mixture of either 90o H20...

The DNA-binding domain of c-Myb consists of three homologous tandem repeats of 52 amino acids. The structure of the third (C-terminal) repeat obtained by NMR analysis has a conformation related to the helix-turn-helix motif. To identify the role of each repeat in the sequence recognition of DNA, we analyzed specific interactions between c-Myb and DNA by measuring binding affinities for systematic mutants of Myb-binding DNA sites and various truncated c-Myb mutants. We found that specific interactions are localized unevenly in the AACTGAC region in the consensus binding site of c-Myb: The first adenine, third cytosine, and fifth guanine are involved in very specific interactions, in which any base substitutions reduce the binding affinity by >500-fold. On the other hand, the interaction at the second adenine is less specific, with the affinity reduction in the range of 6-to 15-fold. The seventh cytosine involves a rather peculiar interaction, in which only guanine substitution abolishes the specific binding. The binding analyses, together with the chemical protection analyses, showed that the c-Myb fragment containing the second and third repeats covers the AACTGAC region from the major groove of DNA in such an orientation that the third repeat covers the core AAC sequence. These results suggest that the third repeat recognizes the core AAC sequence very specifically, whereas the second repeat recognizes the GAC sequence in a more redundant manner. The first (Nterminal) repeat, which covers the major groove of DNA only partially, is not significant in the sequence recognition, but it contributes to increase the stability of the Myb-DNA complex. The presence of an N-terminal acidic region upstream of the first repeat, which is important for the activation of c-myb protooncogene, was found to reduce the binding affinity by interfering with the first repeat in binding to DNA.The protooncogene c-myb codes for the nuclear protein (c-Myb) that binds to DNA in a sequence-specific manner (1, 2). The c-Myb protein is supposed to function as an activator or repressor oftranscription (3-6). The DNA-binding domain of c-Myb consists of three homologous tandem repeats of 52 amino acids [repeat 1 (Ri), repeat 2 (R2), and repeat 3 (R3) from the N terminus] (7-9). Each repeat has three conserved tryptophans spaced 18-19 aa apart (10). Ri can be deleted without significant loss of DNA-binding activity (9, 11). Thus, Rl is thought to be a minor player in sequence recognition. The solution structure of R3 has been obtained by NMR analysis (12). The analysis showed that the conserved tryptophans form a hydrophobic core, as predicted from sequence and mutagenesis analyses (13,14), and that the a-helices fold into a conformation related to the helixturn-helix (HTH) motif. The model of Myb-DNA complexThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.suggests that one of th...

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Expression of the Escherichia coli outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity. The OmpR and EnvZ proteins are transcriptional factors involved in this osmotic regulation of the ompC and ompF genes. In particular, expression of the ompC gene is activated by the positive regulator, OmpR, in response to high osmolarity of the medium. In this study, we succeeded in defining a functional OmpR-binding sequence by analyzing a set of synthetic oligonucleotides, and propose a consensus motif for OmpR-binding. It was also demonstrated that the asymmetric OmpR-binding sequence, thus identified, can activate the canonical ompC promoter in an orientation independent-manner, providing that this sequence is placed closely and stereo-specifically with respect to the -35 region.

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The DNA-binding domain of the c-myb protooncogene product consists of three homologous tandem repeats of 51-52 amino acids (denoted as R1, R2, and R3 from the N-terminal side). In order to analyze conformational and thermodynamic characteristics of the homologous repeats, we have examined the DNA-binding domain by circular dichroism (CD) and differential scanning calorimetry (DSC). The CD spectra for the three individual repeats are significantly different in the fine profiles, indicating subtle differences in their conformations. The melting analyses for the fragments show that the thermal stability of each fragment is different from one another, with the following order of stability: R1(Tm = 61 degrees C) approximately greater than R3(57 degrees C) >> R2(43 degrees C), where R2 is much less stable than the other repeats. The denaturing process for the whole DNA-binding domain, measured by DSC, is characterized by a very broad transition ranging from 30 to 80 degrees C. The denaturation curve can be fit well by a three-state transition with one intermediate state. The transition temperature for the native-to-intermediate transition coincides with the melting temperature of R2, indicating that the intermediate state corresponds to the unfolding of unstable R2. The CD spectrum of the whole domain is almost identical to the sum of the individual spectra. Thus, these results suggest that the individual repeats in the whole DNA-binding domain behave independently in terms of conformation and stability. The addition of DNA to the DNA-binding fragment drastically changed the melting profile, in which the broad transition curve was replaced by a sharp peak at 58 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Fas is a cell-surface receptor that belongs to the tumor-necrosis factor (TNF)/nerve growth factor receptor family. Fas can transduce an apoptotic signal through the death domain in the cytoplasmic region, which has similarity with the corresponding region of the TNF type-I receptor. Here, we expressed human Fas in mouse L929 cells or its subline (C12), which express extremely low levels of cytosolic phospholipase A, (cPLA,). L929 cells were sensitive to the cytotoxic activity of TNF, while C12 cells were resistant. Cross-linking of human Fas with anti-(human Fas antibody) Ig killed both L929 transformants and C12 transformants expressing human Fas. Various inhibitors of the arachidonate metabolism significantly inhibited the TNF-induced cytotoxicity in L929 cells, but they did not have any effect on Fasmediated apoptosis. These results indicated that cPLA, is required for TNF-induced apoptosis, whereas it is dispensable for Fas-mediated apoptosis, and suggested that the TNF receptor and Fas use different signaling pathways for apoptosis.

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