title: In vivo recognition of the fecA3 target promoter by Helicobacter pylori NikR.
abstract: In Helicobacter pylori, the transcriptional regulator HpNikR represses transcription of the fecA3 gene by binding to two adjacent operators spanning a region of almost 80 nucleotides along the fecA3 promoter in a nickel-dependent manner. By employing hydroxyl radical footprinting, we mapped the protected nucleotides within each operator. Three short sequences rich in A and T nucleotides were identified within each operator, comprising just 24 bases for both operators, with 4 or 5 protected bases interspaced by 4 to 7 free nucleotides, with no center of symmetry. Base substitutions at any site strongly reduced the affinity of HpNikR for the operators and also affected the stability of the DNA-protein complex, when the promoter-regulator interaction was analyzed in vitro. The effect of these substitutions was remarkably different when transcription of the mutant promoters was analyzed in vivo. Base changes introduced at the farthest subsites impaired the HpNikR-dependent repression, with the mutations closer to +1 completely abolishing the repression, the more distal one still allowing almost 50% of transcription, and the mutations in the middle being ineffective. The data presented here show that HpNikR may first select its targets by identifying sequences within the previously defined consensus and subsequently establish base-specific contacts to firmly bind DNA. In particular, HpNikR seems to interact in an asymmetric mode with the fecA3 target to repress its transcription.
authors.affiliation: Department of Biology, University of Bologna, Bologna, Italy. vincenzo.scarlato@unibo.it
authors.forename: Simona
authors.initials: S
authors.lastname: Romagnoli
authors.forename: Francesca
authors.initials: F
authors.lastname: Agriesti
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Scarlato
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C523724:NikR protein, Helicobacter pylori; D012097:Repressor Proteins; D017665:Hydroxyl Radical