Recognition of specific DNA sequences by the c-myb protooncogene product: role of three repeat units in the DNA-binding domain.

Abstract

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...

A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene.

“…Second, CCAl probes can only detect a single gene on a DNA gel blot even at low stringency and using a probe including the myb-related sequence. In addition, the DNA region to which it binds, and which is conserved in a number of plant Lhcb genes, does not include the plant consensus sequence CCT/AACC for recognition by the maize Myb P protein (Grotewold et al, 1994), the recognition sequence for other plant Myb proteins (e.g., see Solano et al, 1995), or consensus sequences determined for Myb proteins from other kingdoms (TAACTG) (Tanikawa et al, 1993). We conclude, therefore, that CCAl is a nove1 myb-related gene rather than a member of the myb gene family in Arabidopsis.…”

Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry

“…The dissociation constant in solution (100 mM potassium phosphate buffer (pH 7.5) with 20 mM KCl, 0.1 mM EDTA, 500 g/mL of bovine serum albumin, 5% (vol/ vol) glycerol and 10 mM dithiothreitol) of this complex had been determined as Kd ϭ 3.8 ϫ 10 Ϫ9 M by filter-binding assay [10]. …”

“…The size of target DNA is also important for binding of c-Myb. The c-Myb R1R2R3 bound to the 22-mer, 19-mer, and 16-mer dsDNA fragments in a sequence specific manner with similar affinities, but for the 13-mer and 10-mer fragments, binding affinity was greatly reduced from 10 Ϫ9 M to 10 Ϫ7 M order, even though it contains specific sequence of AACNG [10].…”

“…Two tandem repeats, R2 and R3, are essential and sufficient for the sequence specific-binding of c-Myb but the first repeat, R1, is not necessary for the sequence specific recognition [12], so the minimal DNA-binding domain (DBD) is an R2R3 fragment. The recognition of dsDNA sequence by c-Myb has been studied extensively by NMR, filter-binding assay, mutation analyses, and other biological assays [10,11]. The dissociation constant Kd values of protein-DNA complexes of the minimal and full DNA-binding domain of c-Myb, R1R2, and R1R2R3, with several 16-and 22-mer dsDNA oligomers, have been determined by filter-binding as-say using 32 P-labeled dsDNA.…”

Human A‐myb gene encodes a transcriptional activator containing the negative regulatory domains

“…The c-myb gene product (c-Myb) is a transcriptional activator that can bind to specific DNA sequences, AACNGN(A/T/ C) [2,39,45]. Both artificial promoter constructs containing Myb-binding sites [26,30,44] transcriptional activation, and negative regulation, respectively [35].…”