title: Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion.
abstract: The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. The regulation of the nrd operon has been observed to be very complex. The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described. A partially purified protein from an E. coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990). We have purified this protein to homogeneity by affinity chromatography and identified it as the E. coli factor for inversion stimulation (Fis). Cu-phenanthroline footprinting experiments showed that Fis binds to a site centered 156 bp upstream of the start of nrd transcription. Mutants with deletion and site-directed mutations that do not bind Fis at this site have two- to threefold-lower expression of an nrd-lac fusion. The previously reported negative regulatory nature of this site (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990) was found to be due to a change in polarity in the vectors used to construct promoter fusions. Two nine-base sequences with homology to the DnaA consensus binding sequence are located immediately upstream of the nrd putative -35 RNA polymerase binding site. Binding of DnaA to these sequences on DNA fragments containing the nrd promoter region was confirmed by in vitro Cu-phenanthroline footprinting. Footprinting experiments on fragments with each as well as both of the mutated 9-mers suggests cooperativity between the two sites in binding DnaA. Assay of in vivo expression from wild-type and DnaA box-mutated nrd promoter fragments fused to lacZ on single-copy plasmids indicates a positive effect of DnaA binding on expression of nrd.
authors.affiliation: Department of Biochemistry, University of Minnesota, St. Paul 55108.
authors.forename: L B
authors.initials: LB
authors.lastname: Augustin
authors.forename: B A
authors.initials: BA
authors.lastname: Jacobson
authors.forename: J A
authors.initials: JA
authors.lastname: Fuchs
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D015964:Gene Expression Regulation, Bacterial; D034982:Integration Host Factors; D007763:Lac Operon; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012262:Ribonucleoside Diphosphate Reductase / Q000096:biosynthesis / Q000235:genetics*; D017421:Sequence Analysis
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D011993:Recombinant Fusion Proteins; C440502:integration host factor, E coli; D012262:Ribonucleoside Diphosphate Reductase