RESUMO
Bacterial MerR proteins are dimeric DNA-binding proteins that mediate the Hg(II)-dependent induction of mercury resistance operons. Site-directed mutagenesis of the Bacillus sp. RC607 MerR protein reveals that three of four Cys residues per monomer are required for Hg(II) binding at the single high-affinity binding site. Inactive mutant homodimers can exchange subunits to form heterodimers active for Hg(II) binding. Studies of a heterodimer retaining only three of eight cysteine residues per dimer reveal that Cys79 in one subunit and Cys114 and Cys123 in the second subunit are necessary and sufficient for high-affinity Hg(II) binding in an asymmetric, subunit bridging coordination complex.
Assuntos
Bacillus/análise , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mercúrio/metabolismo , Sequência de Aminoácidos , Bacillus/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Cátions , Proteínas de Ligação a DNA/genética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , Relação Estrutura-AtividadeRESUMO
The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.
Assuntos
Desoxirribonuclease EcoRI/genética , Escherichia coli/genética , Rhodobacter sphaeroides/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon/genética , Escherichia coli/enzimologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Rhodobacter sphaeroides/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.
