Promoter properties and negative regulation of the uvrA gene by the LexA repressor and its amino-terminal DNA binding domain.
A comparative study of the interaction of the LexA repressor of Escherichia coli and of its amino-terminal DNA binding domain to the uvrA operator has been undertaken. Most of the binding constants are determined from competition experiments with RNA polymerase by measuring the time-course of the abortive initiation transcriptional activity. The presence of repressor increases the lag time, tau, without affecting the final maximum activity. The inhibition of transcription by LexA, at least in the case of the uvrA gene, is thus a transient, time-dependent phenomenon, because once the RNA polymerase is engaged in a stable "open" complex, it is quasi-irreversibly trapped in this state. A study of the binding constants as a function of ionic strength suggests the formation of 5.5(+/- 1) salt bridges between the uvrA operator and a LexA dimer. Surprisingly, the binding affinity of the amino-terminal domain was only about one order of magnitude smaller than that of the entire LexA repressor. The determination of the binding constant of the RNA polymerase to the "closed" uvrA promoter (KB approximately 1 X 10(7) to 2 X 10(7) M-1) allowed us to determine theoretical repression curves for the two repressor species. These calculations show that the binding constant found for LexA is sufficiently high to account for substantial or complete repression, and that of the amino-terminal domain is sufficiently low to account for partial or nearly full induction. Under solvent conditions used by others for the determination of binding constants to other SOS operators by DNAase I footprinting, the uvrA operator turns out to be a rather weak one (K approximately 3 X 10(7) M-1), being comparable with that of the uvrB gene. The uvrA promoter is "association-limited" with a KB X k2 product fitting very nicely the homology score for the promoter of 55.
Bertrand-Burggraf
E
E
Hurstel
S
S
Daune
M
M
Schnarr
M
M
D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007700:Kinetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D003850:Deoxyribonuclease I; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein