Superoxide dismutases (SODs) are widely distributed enzymes that convert superoxides to hydrogen peroxide and molecular oxygen, using various metals as cofactors. Many actinobacteria contain genes for both Ni-containing (sodN) and Fe-containing (sodF) SODs. In Streptomyces coelicolor, expression of the sodF and sodN genes is inversely regulated by nickel-specific Nur, a Fur-family regulator. With sufficient nickel, Nur directly represses sodF transcription, while inducing sodN indirectly. Bioinformatic search revealed that a conserved 19-nt stretch upstream of sodN matches perfectly with the sodF downstream sequence. We found that the sodF gene produced a stable small-sized RNA species (s-SodF) of ∼90 nt that harbors the anti-sodN sequence complementary to sodN mRNA from the 5′-end up to the ribosome binding site. Absence of nearby promoters and sensitivity to 5′-phosphate-specific exonuclease indicated that the s-SodF RNA is a likely processed product of sodF mRNA. The s-SodF RNA caused a significant decrease in the half-life of the sodN mRNA. Therefore, Nur activates sodN expression through inhibiting the synthesis of sodF mRNA, from which inhibitory s-SodF RNA is generated. This reveals a novel mechanism by which antagonistic regulation of one gene is achieved by small RNA processed from the 3′UTR of another gene’s mRNA.

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K33FEHH37FEEC41SPC44LEK47 that encompass the conserved HX3CX2C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.

No abstract

Certain bacterial zinc-containing anti-sigma (ZAS) factors respond sensitively to thiol-induced oxidative stress by undergoing conformational changes, which in turn reduce binding affinities for their cognate sigma factors. This redox sensitivity provides a mechanism for coping with oxidative stress by activating the transcription of antioxidant genes. Not all ZAS proteins are redox-sensitive, but the mechanism of redox sensitivity is not fully understood. Here we propose that alternative zinc-binding sites determine redox sensitivity. To support this proposal, we performed protein modeling and zinc docking on redox-sensitive and redox-insensitive ZAS proteins complexed with their cognate sigma factors. At least one strong alternative zinc-binding pocket was detected for all known redox-sensitive ZAS factors in actinomycetes, while no strong alternative zinc-binding pocket was identified in redox-insensitive ZAS factors, except for one controversial case. This hypothesis of alternative zinc-binding sites can also explain residue-specific contributions to the redox sensitivity of RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor, for which alanine mutagenesis experiments are available. Our results suggest a mechanistic model for redox sensitivity as follows: zinc ion can probabilistically occupy multiple sites in redox-sensitive ZAS proteins, increasing the susceptibility of zinc-coordinating cysteine residues to oxidation. This picture of probabilistic zinc occupation agrees with a previous structure and energy analysis on zinc finger proteins, and thus it may be more widely applicable to other classes of reactive zinc-binding proteins.