FNR-DNA interactions at natural and semi-synthetic promoters.
Two rapid and convenient methods have been developed for the amplification and purification of FNR, the anaerobic transcription regulator of Escherichia coli. The overproduced proteins resemble wild-type FNR in their basic properties: oligomeric state, iron contents (up to 2.7 atoms per monomer), DNA-binding affinities and ability to activate transcription. However, unlike previous preparations, FNR could be isolated in a form containing up to 0.25 atoms of acid-labile sulphur per monomer. Incorporation of iron increased the Mr of FNR from 28,000 to 40,000. Under anaerobic conditions, reconstituted FNR exhibited absorption maxima at 315 nm and 420 nm, which were replaced by a broad absorbance from 380 to 440 nm under aerobic conditions. These observations indicate that FNR contains one redox-sensitive [3Fe 4S] or [4Fe 4S] centre per monomer. Footprints of FNR-dependent promoters (ansB, fdn, fnr, narG, pflP6, pflP7 and nirB) showed protection at all of the predicted FNR sites except the pflP7 (-57.5), ansB (-74.5) and nirB (-89.5) sites. An unpredicted second binding site was detected at -57.5 in the narG promoter. Hypersensitive sites within regions of FNR protection indicated that FNR bends DNA in a similar way to CRP. Promoters containing binding sites for FNR (FF), CRP (CC) or hybrid sites (CF or FC) were footprinted with FNR and two derivatives (FNR-610 and FNR-573) which activate the CCmelR promoter in vivo. FNR preferentially protected the FNR site (FF) whereas FNR-610 preferred CC and FNR-573 interacted with equal affinity at all sites.
Green
J
J
Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Irvine
A S
AS
Meng
W
W
Guest
J R
JR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002850:Chromatography, Gel; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017930:Genes, Reporter / Q000235:genetics; D005838:Genotype; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011487:Protein Conformation; D013053:Spectrophotometry; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors