Sequence-specific DNA recognition of the Escherichia coli Ada protein associated with the methylation-dependent functional switch for transcriptional regulation.
The Escherichia coli Ada protein, a suicidal DNA methyltransferase, is converted into a transcriptional regulator for methylation-resistance genes by the transfer of a methyl group from a DNA methylphosphotriester to its own Cys69 residue. Here, we report the DNA recognition mode and the functional switch mechanism of the N-terminal 16 kDa fragment of the Ada protein. NMR analysis has revealed that the segment from residues 102 to 123 forms a helix-turn-helix structure. A site-directed mutagenesis study has shown that the second helix in the helix-turn-helix structure plays a crucial role in specific recognition of DNA. These results imply that the sequence-specific interaction of the Ada protein with DNA occurs through the helix-turn-helix motif. NMR experiments on the methylated protein-DNA complex showed line broadening for the amide proton signals from the helix-turn-helix motif and for the protons in the vicinity of Cys69. In the case of the nonmethylated protein-DNA complex, signal broadening was observed only for protons from the helix-turn-helix. These findings suggest that the residues in the vicinity of Cys69 come into direct contact with the cognate DNA after methylation. We propose that the direct contact of this region is a major factor for the "switch" that converts the Ada protein from a nonspecific DNA binding form to a transcription factor.
Sakashita
H
H
Protein Engineering Research Institute, Osaka.
Sakuma
T
T
Akitomo
Y
Y
Ohkubo
T
T
Kainosho
M
M
Sekiguchi
M
M
Morikawa
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D019077:Helix-Turn-Helix Motifs; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine