RESUMO
The yeast BAS1 protein is a transcriptional activator with an amino-terminal domain homologous to the DNA-binding domain of the oncoprotein Myb containing three imperfect tryptophan-rich repeats. In contrast to Myb-related transcription factors from higher eukaryotes, where the second and third repeat constitutes a minimal independent DNA-binding domain, all three repeats of BAS1 were found to be necessary for sequence-specific DNA binding. Moreover, an active DNA-binding subdomain was obtained only if the first repeat was enlarged in the amino-terminal direction to include 3 tryptophans and a 23-amino acid insertion and if 55 amino acids carboxyl-terminal to the third repeat were included. The BAS1 DNA-binding site was analyzed in detail and found to cover 8-9 base pairs with no similarity to the Myb recognition element. The binding site included a conserved hexameric TGACTC motif, the methylation of which abolished BAS1 binding, as well as a 3-base pair extension that seemed to have a modulatory effect on BAS1 affinity and where binding was less affected by methylation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Galinhas , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-myb , Alinhamento de Sequência , Transativadores/genética , Triptofano/metabolismoRESUMO
The oncoprotein Myb is a sequence-specific DNA-binding protein with a pivotal function in the development and proliferation of hematopoietic precursor cells. A minimal DNA-binding domain composed of two tryptophan-rich repeats R2 and R3 is responsible for sequence recognition. Based on model building and mutational analysis, Myb was proposed to recognise its target through a double helix-turn-helix (HTH)-related motif using two recognition helices, one in R2 and one in R3. We found, by mutational analysis, that the DNA-binding site for c-Myb is functionally bipartite. While the first half site is dominant and absolutely required for binding, the second half site is only modulatory and mainly affects the half life of the complex. This bipartite nature of the binding site parallels the proposed bipartite structure of R2R3 with two HTH-related domains. Analysis of the DNA-binding site of R2R3 by missing-base interference-footprint analysis showed that the protein interacted with a 9-bp region. The same was found with a larger protein containing all three repeats. The effect of adding R1 was mainly to stabilise the complex. The borders of the complex, as revealed by exonuclease III footprinting, did not change due to the presence of R1. However, both borders became more refractory to the nuclease when R1 was present, but with a difference that suggested a specific orientation of the repeat domains relative to the DNA-binding site. We propose that the first half site is recognised by R3, while the second modulatory half site interacts with the R2 repeat.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Galinhas , DNA/genética , Sequências Hélice-Alça-Hélice , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , Proteínas Proto-Oncogênicas c-myb , Proteínas Recombinantes/metabolismoRESUMO
The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2.