Gene activation by the AraC protein can be inhibited by DNA looping between AraC and a LexA repressor that interacts with AraC: possible applications as a two-hybrid system.
The Escherichia coli activator and repressor proteins AraC and LexA bind DNA as homodimers. Here we show that their heterodimerization through fused cognate dimerization domains results in repression of AraC-dependent gene activation by LexA. Repression also requires a LexA operator half-site located several helical turns downstream of the AraC operator. This requirement for a specific spatial organization of the operators suggests the formation of a DNA loop between operator-bound Ara/LexA heterodimers, and we propose that heterodimerization with the AraC hybrid provides co-operativity for operator binding and repression by the LexA hybrid. Consistent with a mechanism that involves DNA looping, repression increases when the E. coli DNA looping and transcriptional effector protein IHF binds between the AraC and LexA operators. Thus, we have combined the functions of three distinct transcriptional effector proteins to achieve a new mode of gene regulation by DNA looping, in which the activator protein is an essential part of the repressor complex. The flexibility of the DNA loop may facilitate this novel combinatorial arrangement of those proteins on the DNA. The requirement for protein interactions between the AraC and LexA hybrids for gene regulation suggests that this regulatory circuit may prove useful as an E. coli-based two-hybrid system.
Kornacker
M G
MG
Department of Macromolecular Structure, Bristol-Myers Squibb Pharmaceutical Research Institute, Route 206 and Province Line Road, Princeton, NJ 08543-4000, USA. KornackM@BMS.com
Remsburg
B
B
Menzel
R
R
D051777:AraC Transcription Factor; D001426:Bacterial Proteins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D016762:Genes, fos / Q000235:genetics; D016758:Genes, jun / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012097:Repressor Proteins / Q000494:pharmacology*; D012697:Serine Endopeptidases; D014157:Transcription Factors; D015533:Transcriptional Activation; D000099160:LexA Repressor Protein
D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C497371:AraC protein, E coli