In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain.
Both the amino-terminal and the carboxy-terminal domain of the LexA repressor have been purified using the LexA protein autodigestion reaction at alkaline pH, which leads to the same specific products as the physiological RecA-catalyzed proteolysis of repressor. We show by circular dichroism (c.d) that, upon non-specific binding to DNA, the purified amino-terminal domain induces a very similar if not identical conformational change of the DNA as does the entire repressor. The positive c.d. signal increases approximately 3-fold if the DNA lattice is fully saturated with protein. Further, the amino-terminal domain of the LexA protein binds specifically to the operator of the recA gene, producing qualitatively the same effects on the methylation pattern of the guanine bases by dimethylsulfate as the entire repressor, consisting of a methylation inhibition effect at four distal operator guanines and a slight enhancement at the central bases. The spacing between these contacts suggests that LexA does not bind to the operator along the same face of the DNA helix. As shown by c.d. studies the amino-terminal domain harbours a substantial amount of residues in alpha-helical conformation, a prerequisite for DNA recognition via a helix--turn--helix structural motif as proposed for many other regulatory proteins.
Hurstel
S
S
Granger-Schnarr
M
M
Daune
M
M
Schnarr
M
M
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001482:Base Composition; D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000378:metabolism*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010446:Peptide Fragments; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein