Mapping arm-DNA-binding domain interactions in AraC.
AraC protein, the regulator of the l-arabinose operon in Escherichia coli has been postulated to function by a light switch mechanism. According to this mechanism, it should be possible to find mutations in the DNA-binding domain of AraC that result in weaker arm-DNA-binding domain interactions and which make the protein constitutive, that is, it no longer requires arabinose to activate transcription. We isolated such mutations by randomizing three contiguous leucine residues in the DNA-binding domain, and then by systematically scanning surface residues of the DNA-binding domain with alanine and glutamic acid. As a result, a total of 20 constitutive mutations were found at ten different positions. They form a contiguous trail on the DNA-distal face of the DNA-binding domain, and likely define the region where the N-terminal arm that extends from the N-terminal dimerization domain contacts the C-terminal DNA-binding domain.
Wu
M
M
Biology Department, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD, 21218, USA.
Schleif
R
R
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019747:Aldose-Ketose Isomerases / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D051777:AraC Transcription Factor; D001089:Arabinose / Q000494:pharmacology; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005643:Fucose / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D005643:Fucose; D018698:Glutamic Acid; D004247:DNA; D001089:Arabinose; D012321:DNA-Directed RNA Polymerases; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; D000409:Alanine