Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region.
LuxR, the Vibrio fischeri luminescence gene (lux) activator, is the best-studied member of a family of bacterial transcription factors required for cell density-dependent expression of specific genes involved in associations with eukaryotic hosts. Neither LuxR nor any other LuxR homolog has been shown to bind DNA directly. We have purified the LuxR C-terminal transcriptional activator domain from extracts of recombinant Escherichia coli in which this polypeptide was expressed. The purified polypeptide by itself binds to lux regulatory DNA upstream of the lux box, a 20-bp palindrome that is required for LuxR activity in vivo, but it does not bind to the lux box. However, the LuxR C-terminal domain together with RNA polymerase protects a region including the lux box and the lux operon promoter from DNase I cleavage. There is very little protection of the lux operon promoter region from DNase I digestion in the presence of RNA polymerase alone. Apparently, there is a synergistic binding of the LuxR C-terminal domain and RNA polymerase to the promoter region. The upstream binding region for the purified polypeptide encompasses a binding site for cAMP receptor protein (CRP). Under some conditions, CRP binding can block the binding of the LuxR C-terminal domain to the upstream binding region, and it can also block the synergistic binding of the LuxR C-terminal domain and RNA polymerase to the lux box and luminescence gene promoter region. This description of DNA binding by the LuxR C-terminal domain should lead to an understanding of the molecular interactions of the LuxR family of transcriptional activators with regulatory DNA.
Stevens
A M
AM
Department of Microbiology, University of Iowa, Iowa City 52242.
Dolan
K M
KM
Greenberg
E P
EP
D001426:Bacterial Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics; D009876:Operon; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011953:Receptors, Cyclic AMP / Q000378:metabolism; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D015534:Trans-Activators; D014733:Vibrio / Q000235:genetics*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011953:Receptors, Cyclic AMP; D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C064778:LuxR autoinducer binding proteins; D012321:DNA-Directed RNA Polymerases