No abstract

No abstract

Sequence analysis of 236 promoters recognized by the Bacillus subtilis sigma A-RNA polymerase reveals an extended promoter structure. The most highly conserved bases include the -35 and -10 hexanucleotide core elements and a TG dinucleotide at position -15, -14. In addition, several weakly conserved A and T residues are present upstream of the -35 region. Analysis of dinucleotide composition reveals A2- and T2-rich sequences in the upstream promoter region (-36 to -70) which are phased with the DNA helix: An tracts are common near -43, -54 and -65; Tn tracts predominate at the intervening positions. When compared with larger regions of the genome, upstream promoter regions have an excess of An and Tn sequences for n > 4. These data indicate that an RNA polymerase binding site affects DNA sequence as far upstream as -70. This sequence conservation is discussed in light of recent evidence that the alpha subunits of the polymerase core bind DNA and that the promoter may wrap around RNA polymerase.

Summary Gene regulation in cis by riboswitches is prevalent in bacteria. The yybP-ykoY riboswitch family is quite widespread, yet its ligand and function remained unknown. Here we characterize the Lactococcus lactis yybP-ykoY riboswitch as a Mn2+-dependent transcription-ON riboswitch, with a ~30–40 μM affinity for Mn2+. We further determined its crystal structure at 2.7 Å to elucidate the metal sensing mechanism. The riboswitch resembles a hairpin, with two coaxially stacked helices tethered by a four-way junction and a tertiary docking interface. The Mn2+-sensing region, strategically located at the highly conserved docking interface, has two metal binding sites. Whereas the one site tolerates binding of both Mg2+ and Mn2+, the other site strongly prefers Mn2+ due to a direct contact from the N7 of an invariable adenosine. Mutagenesis and a Mn2+-free E. coli yybP-ykoY structure further reveal that Mn2+ binding is coupled with stabilization of the Mn2+-sensing region and the aptamer domain.

In Bacillus subtilis, hydrogen peroxide induces the synthesis of catalase (KatA), alkyl hydroperoxide reductase (AhpCF), and a DNA-binding protein of the Dps family (MrgA). KatA, AhpCF, heme biosynthesis enzymes, and MrgA are also induced upon entry into stationary phase under conditions of iron and manganese limitation. In an effort to define the peroxide regulon repressor, PerR, we used mini-Tn10 mutagenesis to identify loci affecting the regulation of mrgA. From this screen, we isolated two mini-Tn10 insertions in ahpC, the gene encoding the small subunit of AhpCF, that increase the transcription of mrgA-lacZ even in ironsupplemented minimal medium. Indeed, these ahpC::Tn10 insertions lead to elevated expression from all peroxide regulon promoters, including those for mrgA, katA, hemAXCDBL, and ahpCF. As a result, the ahpC::Tn10 mutants display an increased resistance to H 2 O 2 . The ahpCF promoter region contains three sequences similar to the peroxide regulon consensus operator (per box). We demonstrate that the ability of ahpC::Tn10 mutations to derepress mrgA requires aerobic growth. In contrast, a second distinct trans-acting regulatory mutation bypasses this requirement for aerobic growth. Since the peroxide regulon is activated in the absence of AhpCF, which degrades alkyl hydroperoxides, we propose that organic hydroperoxides may be physiologically relevant inducers in vivo.