IadR (Vapar_1489, also designated MarR_73) is a 158-amino-acid indole-3-acetic acid (IAA)-sensing MarR-family DNA-binding transcriptional repressor that controls expression of the iad auxin-catabolic operon in Variovorax paradoxus S110. IadR forms a homodimer containing a winged-helix-turn-helix (wHTH) DNA-binding domain (residues 13-145) with an H-T-H motif (residues 59-82) and binds specifically to a 24 bp operator site in the intergenic region between iadR and iadA to repress transcription of the iad operon in the absence of auxin. When IAA is present, each IadR monomer binds IAA directly with sub-micromolar affinity (Kd = 0.41 uM) via an indole-binding pocket (Tyr15, Ala18, Trp45, Arg46, Val65) and carboxylate-coordinating triad (Ser28, His32, Arg46), triggering a conformational shift in the DNA-binding helices that releases IadR from operator DNA and derepresses transcription of the IAA degradation pathway.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000104 | MODIFY | Summary: IadR binds specifically to a 24 bp double-stranded DNA operator between iadR and iadA. Reason: The general term DNA binding (GO:0003677) should be refined to transcription cis-regulatory region binding (GO:0000976), as ITC and EMSA experiments demonstrate sequence-specific binding to the 24 bp operator upstream of the iad operon. Proposed replacements: transcription cis-regulatory region binding Supporting Evidence: PMID:36266335 Using MarR_73 S28A protein with reduced ligand binding capacity (Supplementary Table 2), we identified a 24βbp double-stranded DNA oligo carrying a sequence located between MarR_73 (iadR) and gene 72 (iadA) that was capable of binding to this variant MarR_73 protein (Fig. 2d). |
| GO:0003700 DNA-binding transcription factor activity | IEA GO_REF:0000002 | MODIFY | Summary: IadR functions as an IAA-inactivated DNA-binding transcriptional repressor of the iad operon. Reason: DNA-binding transcription factor activity (GO:0003700) is true but less specific than DNA-binding transcription repressor activity (GO:0001217), which is established by ΞMarR_73 RNA-seq and operator-binding assays. Proposed replacements: DNA-binding transcription repressor activity Supporting Evidence: PMID:36266335 These data (Fig. 2) demonstrate that MarR_73 functions as a repressor of this locus: in the absence of IAA, MarR_73 binds to a DNA site between MarR_73 (iadR) and iadA to repress expression of the IAA degradation genes. |
| GO:0006351 DNA-templated transcription | IEA GO_REF:0000104 | REMOVE | Summary: IadR is a transcriptional regulator, not a subunit of the core RNA polymerase machinery. Reason: Per GO curation conventions, transcription factors that regulate gene expression do not directly execute DNA-templated transcription (GO:0006351); IadR negatively regulates transcription (GO:0045892). Supporting Evidence: PMID:36266335 These data (Fig. 2) demonstrate that MarR_73 functions as a repressor of this locus: in the absence of IAA, MarR_73 binds to a DNA site between MarR_73 (iadR) and iadA to repress expression of the IAA degradation genes. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000120 | MODIFY | Summary: IadR negatively regulates transcription of the iad auxin-degradation operon in the absence of IAA. Reason: The general term regulation of DNA-templated transcription (GO:0006355) should be refined to negative regulation of DNA-templated transcription (GO:0045892) based on RNA-seq of the ΞMarR_73 deletion mutant. Proposed replacements: negative regulation of DNA-templated transcription Supporting Evidence: PMID:36266335 In the CL14 ΞMarR_73 mutant, the genes downstream of gene 69 (iadD) are upregulated even in the absence of IAA. This demonstrates the repressive regulatory role of MarR_73 in the absence of IAA. |
| GO:0006950 response to stress | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter over-propagated response to stress from antibiotic/oxidative-stress MarR family members; IadR specifically senses indole-3-acetic acid (IAA) to regulate auxin catabolism. Reason: IadR is a specialized auxin sensor that regulates transcription of the iad indole-3-acetic acid catabolic operon rather than a cellular stress response pathway. Supporting Evidence: PMID:36266335 These data (Fig. 2) demonstrate that MarR_73 functions as a repressor of this locus: in the absence of IAA, MarR_73 binds to a DNA site between MarR_73 (iadR) and iadA to repress expression of the IAA degradation genes. |
| GO:0001217 DNA-binding transcription repressor activity | IDA PMID:36266335 Diverse MarR bacterial regulators of auxin catabolism in the... | NEW | Summary: IadR binds the 24 bp operator upstream of iadA to repress transcription of the iad operon in the absence of IAA. Reason: Demonstrated by operator DNA binding (ITC) and ΞMarR_73 knockout transcriptomics in PMID:36266335. Supporting Evidence: PMID:36266335 These data (Fig. 2) demonstrate that MarR_73 functions as a repressor of this locus: in the absence of IAA, MarR_73 binds to a DNA site between MarR_73 (iadR) and iadA to repress expression of the IAA degradation genes. |
| GO:0000976 transcription cis-regulatory region binding | IDA PMID:36266335 Diverse MarR bacterial regulators of auxin catabolism in the... | NEW | Summary: IadR binds a specific 24 bp palindromic operator sequence in the intergenic regulatory region between iadR and iadA. Reason: Directly measured by isothermal titration calorimetry with the 24 bp double-stranded operator DNA oligonucleotide in PMID:36266335. Supporting Evidence: PMID:36266335 Using MarR_73 S28A protein with reduced ligand binding capacity (Supplementary Table 2), we identified a 24βbp double-stranded DNA oligo carrying a sequence located between MarR_73 (iadR) and gene 72 (iadA) that was capable of binding to this variant MarR_73 protein (Fig. 2d). |
| GO:0045892 negative regulation of DNA-templated transcription | IMP PMID:36266335 Diverse MarR bacterial regulators of auxin catabolism in the... | NEW | Summary: IadR represses transcription of the iad auxin-degradation operon in the absence of IAA. Reason: RNA-seq of the ΞMarR_73 mutant shows constitutive upregulation of the iad operon in the absence of IAA. Supporting Evidence: PMID:36266335 In the CL14 ΞMarR_73 mutant, the genes downstream of gene 69 (iadD) are upregulated even in the absence of IAA. This demonstrates the repressive regulatory role of MarR_73 in the absence of IAA. |
| GO:0005829 cytosol | IC PMID:36266335 Diverse MarR bacterial regulators of auxin catabolism in the... | NEW | Summary: IadR functions in the bacterial cytosol as a soluble dimeric transcriptional repressor. Reason: Inferred by curator (IC) because IadR is a soluble cytoplasmic DNA-binding transcriptional repressor lacking signal peptides or transmembrane helices that senses intracellular IAA. Supporting Evidence: PMID:36266335 These data (Fig. 2) demonstrate that MarR_73 functions as a repressor of this locus: in the absence of IAA, MarR_73 binds to a DNA site between MarR_73 (iadR) and iadA to repress expression of the IAA degradation genes. |
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Download this section (compressed HTML)Q: What is the identity of the additional IAA-responsive transcriptional regulator that further induces the iad operon upon IAA addition in a ΞiadR (ΞMarR_73) mutant background?
Experiment: Determine the co-crystal structure of unliganded IadR bound to its 24 bp operator DNA duplex and compare it against the 1.3 A IAA-bound structure (PDB 7KFO).
Hypothesis: Operator DNA binding stabilizes a ~28 A inter-recognition-helix spacing in unliganded IadR, whereas IAA binding locks the wHTH domains into the 38 A splayed conformation observed in PDB 7KFO.
Type: X-ray crystallography of protein-DNA complex
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