Semisynthetic promoters activated by cyclic AMP receptor protein of Escherichia coli

Abstract

Semisynthetic promoters activated by Escherichia coli cyclic AMP receptor protein (CRP) were created by combining a synthetic CRP-binding site ( crb) and nucleotide sequences derived from cryptic promoter regions. A 22-bp oligodeoxyribonucleotide corresponding to an idealized crb was randomly placed into DNA regions that precede a promoterless lacZ gene on a plasmid. Several plasmid clones were obtained which allowed the expression of lacZ in crp + cya + cells carrying a chromosomal deletion of lac genes. The β-galactosidase and the quantitative S1-nuclease assays of crp + and Δ crp cells harboring these plasmids indicated that the transcription from newly created promoters is dependent on CRP. Sequence analysis revealed that these promoters are divided into two types based on the location of the crb relative to the transcription start point (tsp). The distance from the center of the crb to the tsp is 70 bp in the first type and 38 bp in the second type. The sequences of all these promoters exhibit poor homology with the consensus promoter sequence.