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Studying the Dimerization of the FNR Transcription Factor by Mutant Analysis at the 130 and 140 Residue Positions
Author(s) -
Kazmouz Sobhi,
Moore Laura J.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.792.10
Subject(s) - mutant , biochemistry , escherichia coli , site directed mutagenesis , residue (chemistry) , alanine , salt bridge , chemistry , mutagenesis , dimer , directed mutagenesis , transcription (linguistics) , leucine , biology , amino acid , gene , linguistics , philosophy , organic chemistry
The Fumarate Nitrate Reductase (FNR) transcription factor is the main anaerobic switch in Escherichia coli bacteria. In the absence of oxygen, the FNR protein exists as a dimer with a [4Fe‐4S] cluster and binds to DNA to regulate transcription of genes needed for anaerobic respiration. When oxygen is present, however, the [4Fe‐4S] cluster degrades causing FNR to return to its inactive monomeric form. Examination of the crystal structure of Alivibrio fischeri FNR, a close variant of E. coli FNR, suggested that the dimerization and, thus, activation of FNR is dependent upon a salt bridge interaction between the Asp 130 residue and the Arg 140 residue. [1] Site‐directed mutagenesis was used to analyze how various mutations at those two particular residues can affect FNR activity. Analysis of double mutants where the 130 and 140 residues were swapped showed that the salt bridge interaction may not be playing a role in FNR dimerization since the double mutants had no FNR activity. Therefore, additional mutations were constructed to study the importance of the 130 and 140 residues in promoting dimerization. Data from β – galactosidase assays carried out on an E. coli reporter strain to measure FNR activity suggest that only Arg 140 is essential for regulating dimerization. Single mutants in which Asp 130 was replaced by hydrophobic and polar amino acids display FNR activity almost the same as that of the wild type FNR while the same mutations that replaced Arg 140 resulted in almost no FNR activity. Furthermore, alanine and leucine double mutants at positions 130 and 140 displayed FNR activity even higher than the wild type FNR activity, further suggesting a salt bridge is not required for dimerization. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .