RESUMO
The redox-sensitive OxyR protein activates the transcription of antioxidant defense genes in response to oxidative stress and represses its own expression under both oxidizing and reducing conditions. Previous studies showed that OxyR-binding sites are unusually long with limited sequence similarity. Here, we report that oxidized OxyR recognizes a motif comprised of four ATAGnt elements spaced at 10 bp intervals and contacts these elements in four adjacent major grooves on one face of the DNA helix. In contrast, reduced OxyR contacts two pairs of adjacent major grooves separated by one helical turn. The two modes of binding are essential for OxyR to function as both an activator and a repressor in vivo. We propose that specific DNA recognition by an OxyR tetramer is achieved with four contacts of intermediate affinity allowing OxyR to reposition its DNA contacts and target alternate sets of promoters as the cellular redox state is altered.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fatores de Transcrição , Adaptação Biológica , Sequência de Bases , Sítios de Ligação , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Oxirredução , Oxirredutases/genética , Peroxidases/genética , Peroxirredoxinas , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-Atividade , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.
