Binding of the Bacillus subtilis LexA protein to the SOS operator.
The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5'-CGAACN(4)GTTCG-3'. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5' end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5'-CGAACATATGTTCG-3'. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator.
Groban
Eli S
ES
Department of Chemistry, Williams College, Williamstown, MA 01267, USA.
Johnson
Martha B
MB
Banky
Poopak
P
Burnett
Peta-Gaye G
PG
Calderon
Georgina L
GL
Dwyer
Erica C
EC
Fuller
Shakierah N
SN
Gebre
Biniam
B
King
Leah M
LM
Sheren
Ila N
IN
Von Mutius
Lindi D
LD
O'Gara
Thomas M
TM
Lovett
Charles M
CM
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D009875:Operator Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013014:SOS Response, Genetics; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011938:Rec A Recombinases; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein