Mutational analysis of the main CytR protein binding site within the regulatory region of the Escherichia coli udp gene

Site-specific mutagenesis of the pentameric motif TGCAA within the regulato ry region of the udp gene with coordinates -68 and -64 relative to the tran scription initiation site was performed. Nine mutant promoters containing m ultiple nucleotide base-pair substitutions in this pentameric motif were is olated and characterized. One mutant contained a deletion of the C/G nucleo tide pair in the -66 position. Isolated mutant promoters were cloned into a low-copy-number expression vector pJEL250 to determine the level of their expression, depending on the allelic state of cytR and cya genes. The level of CytR-dependent regulation of the udp gene and the ability to titrate th e CytR repressor in vivo were shown to be drastically decreased in all muta nt promoters isolated. On the basis of these results, it is concluded that the pentameric motif TGCAA plays a key role in binding the CytR repressor p rotein to the udp gene promoter.