Orientation of the LexA DNA-binding motif on operator DNA as inferred from cysteine-mediated phenyl azide crosslinking.
To address the question how the recognition helix of the LexA repressor is positioned within the major groove of operator DNA we have applied a site-specific photocrosslinking approach using a LexA mutant repressor (LexA-C52) that harbors a single cysteine side chain in position 52, close to the COOH terminus of helix 3. The LexA-C52 mutant repressor has been purified and modified site-specifically with the photoreactive azido compound 4-azidophenacyl bromide, giving rise to LexA-C52*. Here we show that LexA-C52* may be selectively photocrosslinked with two adjacent bases within each operator half-site. The crosslinked bases are located, respectively, 10 and 11 base pairs from the dyad axis of the operator. The crosslinking data imply that the LexA recognition helix is oriented opposite to what is generally observed for helix-turn-helix proteins and that this helix should form a steeper angle with respect to the plane of the base pairs than is observed for standard helix-turn-helix proteins.
Dumoulin
P
P
Institut de Biologie Moléculaire et Cellulaire, UPR 9005 du Centre National de la Recherche Scientifique, Strasbourg, France.
Oertel-Buchheit
P
P
Granger-Schnarr
M
M
Schnarr
M
M
D001386:Azides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013014:SOS Response, Genetics; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein
D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein