title: Characterization of specific nucleotide substitutions in DtxR-specific operators of Corynebacterium diphtheriae that dramatically affect DtxR binding, operator function, and promoter strength.
abstract: The diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae uses Fe(2+) 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.
authors.affiliation: Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: R K
authors.initials: RK
authors.lastname: Holmes
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003353:Corynebacterium diphtheriae / Q000235:genetics* / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D035941:Iron Regulatory Protein 1; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D016601:RNA-Binding Proteins; D035941:Iron Regulatory Protein 1