Characterization of specific nucleotide substitutions in DtxR-specific operators of Corynebacterium diphtheriae that dramatically affect DtxR binding, operator function, and promoter strength

The diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae uses Fe²⁺ as a corepressor. Holo-DtxR inhibits transcription from the iron- regulated promoters (IRPS) designated IRP1 through IRP5 as well as from the promoters for the tox and hmuO genes, DtxR binds to 19-bp operators with the consensus sequence 5'-TTAGGTTAGCCTAACCTAA-3', a perfect 9-bp palindrome interrupted by a single C · G base pair. Among the seven known DtxR-specific operators, IRP3 exhibits the weakest binding to DtxR. The message (sense) strand of the IRP3 operator (5'-TTAGGTGAGACGCACCCAT-3' [nonconsensus nucleotides underlined]) overlaps by 2 nucleotides at its 5' end with the putative -10 sequence of the IRP3 promoter. The underlined C at position +7 from the center of the IRP3 operator [C(+7)] is unique, because T is conserved at that position in other DtxR-specific operators. The present study examined the effects of nucleotide substitutions at position +7 or -7 in the IRP3 operator. In gel mobility shift assays, only the change of C(+7) to the consensus nucleotide T caused a dramatic increase in the binding of DtxR, whereas other nucleotide substitutions for C(+7) or replacements for A(-7) had only small positive or negative effects on DtxR binding. All substitutions for C(+7) or A(-7) except for A(-7)C dramatically decreased IRP3 promoter strength. In contrast, the A(-7)C variant caused increased promoter strength at the cost of nearly eliminating repressibility by DtxR. The message (sense) strand of the IRP1 operator (5'-TTAGGTTAGCCAAACCTTT-3') includes the -35 region of the IRP3 promoter, A T(+7)C variant of the IRP1 operator was also constructed, and it was shown to exhibit decreased binding to DtxR, decreased repressibility by DtxR, and increased promoter strength. The nucleotides at positions +7 and -7 in DtxR-specific operators are therefore important determinants of DtxR binding and repressibility of transcription by DtxR, and they also have significant effects on promoter activity for RP3 and IRP1.