Fluorescence study of the RecA-dependent proteolysis of LexA, the repressor of the SOS system in Escherichia coli.
The fluorescence of the LexA protein, the common repressor of the SOS system in Escherichia coli decreases by about 30% upon incubation with the RecA protein, and its cofactors ATP [or its non-hydrolysable analogue adenosine-5'-O-(3-thiotriphosphate), ATP gamma S] Mg2+ and single-stranded DNA. In the absence of any one of these elements required for the RecA-dependent proteolysis of LexA, this fluorescence change was not observed. The final fluorescence change depends only upon the concentration of LexA regardless of that of RecA. The time course of the fluorescence decrease corresponds well with the kinetics of the decrease of intact LexA protein and the increase of its 2 proteolytic fragments as determined by SDS-polyacrylamide gel electrophoresis. These results allow us to use the fluorescence change as a signal for a detailed kinetic analysis. The velocity of the proteolysis (d[LexA]/dt) is proportional to the concentration of LexA and RecA indicating that the formation of the LexA-RecA complex is the limiting step.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D013050:Spectrometry, Fluorescence; D000099160:LexA Repressor Protein
D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D010447:Peptide Hydrolases; D011938:Rec A Recombinases; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein