IDENTIFICATION OF HIGH-AFFINITY BINDING-SITES FOR LEXA WHICH DEFINE NEW DNA DAMAGE-INDUCIBLE GENES IN ESCHERICHIA-COLI

A multi-step screening procedure was devised to identify new operators for the LexA repressor in the sequenced portions of the genomes of Es cherichia coli and its plasmids and bacteriophages. Sequence analysis methods were employed initially to distinguish true LexA operators fro m ''operator-like'' sequences stored within the GenBank and EMBL datab ases. The affinity of purified LexA protein for cloned DNA fragments c ontaining several of the prospective new sites was then assessed using quantitative electrophoretic mobility shift assays and site-directed mutagenesis. Calculated binding affinities were compared directly with values determined for known and mutant LexA operators in concurrent e xperiments. Three E. coli chromosomal segments (near pyrC, hsdS and nt rla) and two bacteriophage sequences (near the P1 cre and lambda oop g enes) bound LexA protein specifically. These sites and most others ide ntified in the screening are located immediately upstream of known gen es and/or large open reading frames. These results and additional tran scription data demonstrate that several of the sequences define new DN A damage-inducible (din) genes and include the previously uncharacteri zed dinD locus. Furthermore, the search identified an SOS gene within the genome of P1 which encodes a protein that is homologous to UmuD', the RecA-promoted cleavage product of the umuD gene. The success of th e combinatorial approach described here suggests that analogous search es for new regulatory sequences within the E. coli genome and the geno mes of other organisms will also yield favorable results.