In vivo genomic footprinting analysis reveals that the complex Bradyrhizobium japonicum fixRnifA promoter region is differently occupied by two distinct RNA polymerase holoenzymes.
The Bradyrhizobium japonicum fixRnifA operon is transcribed from two promoters: fixRp1, a -24/-12 promoter recognized by the sigma54-holoenzyme form of the RNA polymerase, and fixRp2, a -35/-10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The fixRp1 promoter is autoregulated during microaerobiosis by NifA, whereas fixRp2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved -12 and -10 regions of fixRp1 and fixRp2, respectively, must overlap each other, whereas the -24 and -35 regions lie one DNA helical turn apart. Using in vivo genomic dimethyl sulfate and KMnO4 footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from -12 to -14 are critical for the activity of both promoters, whereas mutations at -10 and -11 affected mainly fixRp2 expression. When the sequence of the putative -35 region of fixRp2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO4, but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine -75. The latter residue is located in a region critical for fixRp2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the fixRnifA promoter region.
Barrios
H
H
Departamento de Reconocimiento Molecular y Bioestructura, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, Mexico.
Grande
R
R
Olvera
L
L
Morett
E
E
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D018983:DNA Footprinting; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007536:Isomerism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010088:Oxidoreductases; D011196:Potassium Permanganate / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012230:Rhizobiaceae / Q000235:genetics*; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C061338:NifA protein, Bacteria; D013463:Sulfuric Acid Esters; D014157:Transcription Factors; D011196:Potassium Permanganate; D010088:Oxidoreductases; C092059:FixR protein, Bradyrhizobium japonicum; D012321:DNA-Directed RNA Polymerases; C007482:dimethyl sulfate