Activation from a Distance: Roles of Lrp and Integration Host Factor in Transcriptional Activation of gltBDF

AUTOR(ES)

Paul, Ligi

FONTE

American Society for Microbiology

RESUMO

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 ς70-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 ς70-dependent promoter lacZYAp. The glt-lac promoter hybrids were placed upstream of lacZ, allowing transcriptional activity to be monitored via β-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.

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