Characterization of Specific Nucleotide Substitutions in DtxR-Specific Operators of Corynebacterium diphtheriae That Dramatically Affect DtxR Binding, Operator Function, and Promoter Strength

AUTOR(ES)

Lee, John H.

FONTE

American Society for Microbiology

RESUMO

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 IRP3 and IRP1.

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