Mutants of the lac promoter with large insertions and deletions between the CAP binding site and the −35 region

Abstract

A set of the lac promoter mutants that have varying lengths of the spacer between the CAP binding site and the −35 region was constructed. The mutants have the spacer length increased by five ( I5 mutant), or eleven ( I11) residues or decreased by eleven residues ( D11). We also present a construction of the hybrid between the gal and lac promoters in which the CAP binding site and the −35 region of the gal promoter are fused to the lac −10 region. The promoter fragments were assembled through ligations of chemically synthesized oligodeoxynucleotides and cloned into a pBR322-derivative vector. The results of the in vivo assays of promoter activity show that the I11 mutation results in an active but weak promoter that can be stimulated by CAP, though to a lesser degree than the wild-type lac promoter. The other mutants exhibit no promoter activity. Since the insertion of 11-bp preserves the location of the CAP binding site on the same side on the DNA helix, the data demonstrate the importance of spatial alignment between the CAP binding site and the promoter. The fact that the gal :: lac hybrid is inactive as a promoter indicates also that catabolite activation is a highly complex process in which the −35 and −10 regions cannot be easily exchanged between promoters.