Activation from a distance: roles of Lrp and integration host factor in transcriptional activation of gltBDF.
The leucine-responsive regulatory protein (Lrp) binds to three sites centered 252, 216, and 152 bp upstream of the transcription start site of the Escherichia coli glutamate synthase operon (gltBDF) and activates transcription. Activators of sigma(70)-dependent promoters usually bind closer to the -35 hexamer of the core promoter sequence. To study the mechanism by which Lrp-dependent activation occurs over this relatively large distance, the gltBDF upstream region was sequentially replaced with corresponding portions from the well-characterized sigma(70)-dependent promoter lacZYAp. The glt-lac promoter hybrids were placed upstream of lacZ, allowing transcriptional activity to be monitored via beta-galactosidase assays. Even replacing all gltBDF sequences downstream of and including the -35 hexamer did not eliminate Lrp-dependent activation of transcription. When a 91-bp region between the -35 hexamer and the proximal Lrp binding site (-48 to -128) was replaced with heterologous DNA of the same length, transcription was reduced nearly 40-fold. Based on the presence of a consensus binding sequence, this region seemed likely to be a binding site for integration host factor (IHF). Experiments to study the effects of a himD mutant on expression of a gltB::lacZ transcriptional fusion, gel mobility shift analyses, and DNA footprinting assays were used to confirm the direct participation of IHF in gltBDF promoter regulation. Based on these results, we suggest that IHF plays a crucial architectural role, bringing the distant Lrp complex in close proximity to the promoter-bound RNA polymerase.
Paul
L
L
Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109, USA.
Blumenthal
R M
RM
Matthews
R G
RG
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005970:Glutamate Synthase / Q000235:genetics*; D034982:Integration Host Factors; D051756:Leucine-Responsive Regulatory Protein; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; C497278:Lrp protein, E coli; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein; D005970:Glutamate Synthase