Premium
Investigation of the Molecular Basis of pH‐induced DNA Binding of CooA, a CO‐Sensing Transcription Factor
Author(s) -
Weaver Brian,
Westerlund Geoffrey,
McGhiey Celia,
Markovic Sam,
Clark Robert
Publication year - 2017
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.31.1_supplement.913.19
Subject(s) - heme , rhodospirillum rubrum , effector , dna , transcription factor , chemistry , mutagenesis , electrophoretic mobility shift assay , hemeprotein , transcription (linguistics) , bacterial transcription , biochemistry , microbiology and biotechnology , gene , biology , gene expression , promoter , mutation , enzyme , linguistics , philosophy
The carbon monoxide (CO) sensing heme protein, CooA, is a transcription factor that occurs in several bacteria and regulates the expression of genes, enabling these organisms to utilize CO as a sole energy source. Previous studies by our lab yielded the unexpected result that Fe(III) CooA binds DNA tightly at pH < 7, which deviates from the traditional understanding that CooA DNA binding is initiated only when the exogenous CO effector reacts with the Fe(II) CooA heme. This observation suggests that the disruption of one or more salt bridges upon effector binding may be a critical feature of the normal CooA activation mechanism. To test this possibility, a number of protein variants that eliminate selected salt bridges for the CooA homologs from Rhodospirillum rubrum and Carboxydothermus hydrogenoformans were prepared via site‐directed mutagenesis. Purified samples of these variant proteins, which were overexpressed in Escherichia coli , were then characterized by spectroscopic methods and functional assays to investigate the impact these mutations had on CooA heme coordination structure and DNA‐binding activity. Results of this work are presented in light of the accepted CooA activation mechanism.