title: Requirement for two conserved cysteine residues in the Ada protein of Escherichia coli for transactivation of the ada promoter.
abstract: Cysteine residue 69 of the Escherichia coli Ada transcription factor, which accepts a methyl group from methylphosphotriester in methylated DNA, was substituted by each of 19 other amino acids. Only the mutant Ada (C69H), carrying a histidine substitution of Cys69, exhibited a limited degree of transactivating potential for the ada promoter in E. coli cells although the mutant protein was completely devoid of methylphosphotriester-DNA methyltransferase activity. Using a multicopy plasmid system for the expression of Ada protein, we have shown that Ada C69H has a transactivating capacity equivalent to that of wild-type Ada protein in the absence of an alkylating agent. This indicates that the zinc-binding capacity of histidine at residue 69 is likely to be sufficient for Ada to recognize and bind to the ada promoter. Furthermore, transactivation of the ada promoter by Ada C69H was enhanced up to 6-fold by treatment with methylating agents. An additional substitution was made with alanine in Ada C69H, replacing Cys321, the site for acceptance of a methyl group from O6-methylguanine and O4-methylthymine residues in DNA, with alanine. This renders the protein completely inactive as a methyltransferase but this derivative is constitutively active as a transactivator for the ada promoter. Therefore, acquisition of a methyl group at Cys321 apparently enhances the transactivating capacity of Ada protein on the ada promoter. We propose that the transcription-regulating function of Ada protein is under dual control by methylation of cysteine residues at positions 69 and 321; the former enhances DNA binding, while the latter enhances the transactivating capacity of the protein.
authors.affiliation: Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Taketomi
authors.forename: Y
authors.initials: Y
authors.lastname: Nakabeppu
authors.forename: K
authors.initials: K
authors.lastname: Ihara
authors.forename: D J
authors.initials: DJ
authors.lastname: Hart
authors.forename: M
authors.initials: M
authors.lastname: Furuichi
authors.forename: M
authors.initials: M
authors.lastname: Sekiguchi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017124:Conserved Sequence; D003545:Cysteine; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014157:Transcription Factors; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D014157:Transcription Factors; D006639:Histidine; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine