Mutagenesis of the cysteine residues in the transcription factor NtcA from Anabaena PCC 7120 and its effects on DNA binding in vitro.
NtcA is a transcription factor found in a wide variety of cyanobacteria. It is a key component in the control of the nitrogen metabolism, and regulates genes involved in ammonia assimilation, heterocyst differentiation and nitrogen fixation. NtcA expression is subject to nitrogen control, but there is also evidence that the binding of NtcA to DNA can be regulated by a redox mechanism involving the two cysteine residues in the NtcA protein from Anabaena PCC 7120. In order to investigate this further, the two cysteine residues in NtcA were mutated into alanine to give four variants of the protein: wild-type NtcA, the point-mutated variants Cys157Ala and Cys164Ala, as well as the double mutant Cys157Ala/Cys164Ala. The binding of a DNA probe containing a palindromic NtcA-binding motif was investigated by gel mobility shift analysis under non-reducing and reducing conditions. The experiments show that the DNA binding in vitro is stronger in the presence of the reducing agent DTT than in its absence. However, this effect is not due to breaking of a disulfide bond between the cysteine residues, since the double mutant containing no cysteines was also affected by DTT. A molecular model of a monomer of NtcA, based on the homologous cAMP receptor protein structure, was created in order to locate the positions of the cysteine residues. The NtcA model suggested that the positions of the sulfur atoms are not compatible with formation of a bond between them.
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
Susanne
S
Wisén
Birgitta
B
Bergman
Bengt
B
Mannervik
D017033:Anabaena / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004226:Dithioerythritol; D024202:Electrophoretic Mobility Shift Assay; D005456:Fluorescent Dyes; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D014157:Transcription Factors; C075617:ntcA protein, Synechococcus; D004226:Dithioerythritol; D003545:Cysteine