RESUMO
The Fur (ferric uptake regulation) protein is a global regulator that, in the presence of Fe2+, represses the expression of a number of iron-acquisition genes and virulence determinants such as toxins. Dark-field electron microscopy of positively stained Fur-DNA complexes in addition to atomic force microscopy allowed direct visualization of Fur interactions with the regulatory regions of aerobactin and hemolysin operons and provided complementary information about the structure of the complexes. According to the DNA used and the protein/DNA ratio, Fur binding to DNA results in partial or total covering of the fragments, indicating that the protein initiates polymerization along the DNA molecules at specific sites. Negative staining of Fur-DNA complexes revealed a well-ordered structure of the polymer suggesting a helical arrangement. Local rigidification of the DNA molecules resulting from Fur binding could be involved in the repression process.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Proteínas de Bactérias/química , DNA Bacteriano/química , Ácidos Hidroxâmicos , Ferro/metabolismo , Substâncias Macromoleculares , Microscopia de Força Atômica , Microscopia Eletrônica , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/química , Mapeamento por RestriçãoRESUMO
Fur-DNA interactions were analyzed within the regulatory regions of aerobactin and hemolysin operons by a combination of biochemical and ultrastructural methods. Cartography of the Fur binding sites, carried out from electron micrographs, agreed with the data obtained by DNase I footprinting. Visualization of the complexes confirmed the specificity and metal-dependence of Fur binding and demonstrated that the protein polymerizes on its binding sites. Such a polymerization could be involved in the repression process of the bacterial regulator.