A nucleoprotein activation complex between the leucine-responsive regulatory protein and DNA upstream of the gltBDF operon in Escherichia coli.
The global regulator Lrp (leucine-responsive regulatory protein), in some cases modulated by its co-regulator leucine, has been shown to regulate more than 40 genes and operons in Escherichia coli. Leucine modulates Lrp regulation of leucine-responsive operons. The level of sensitivity of these operons to leucine varies greatly, but the basis for this variation is only partially understood. One operon controlled by Lrp that is relatively insensitive to leucine is gltBDF, which includes genes specifying the large (GltB) and small (GltD) subunits of glutamate synthase. Earlier gel mobility shift assays have demonstrated that Lrp binds to a fragment of DNA containing the gltBDF promoter region. To further define the nature of this Lrp-gltBDF interaction, DNase I footprinting experiments were performed. The results indicate that Lrp binds cooperatively to three sites quite far upstream, spanning the region from -140 to -260 base-pairs relative to the start of transcription. Phased hypersensitivity is observed throughout the entire binding region, suggesting that Lrp bends the DNA. To determine the relative importance of these three sites for the transcriptional activation of gltBDF, a series of site-directed mutations was generated. The effects of these mutations on Lrp binding were determined both by DNase I footprinting and by quantitative mobility shift assays, while their effects on transcription in vivo were examined by measuring beta-galactosidase activity levels of chromosomal gltB::lacZ operon fusions. Our results indicate that all three sites are required for maximal gene expression, as is the proper phasing of the sites with one another and with the start of transcription. Our results suggest that Lrp binds a central palindromic site, interacting predominantly with the major groove of its DNA target, and that additional dimers bind to flanking sites to form a nucleoprotein activation complex.
Wiese
D E
DE
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-1055, USA.
Ernsting
B R
BR
Blumenthal
R M
RM
Matthews
R G
RG
D001426:Bacterial Proteins / Q000235:genetics*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter; D005970:Glutamate Synthase / Q000235:genetics*; D007930:Leucine / Q000235:genetics / Q000494:pharmacology; D051756:Leucine-Responsive Regulatory Protein; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009698:Nucleoproteins / Q000235:genetics*; D009876:Operon; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; D009698:Nucleoproteins; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein; D005970:Glutamate Synthase; D003850:Deoxyribonuclease I; D007930:Leucine