DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter.
The NtrC protein activates transcription of the glnA operon of enteric bacteria by stimulating the formation of stable "open" complexes by RNA polymerase (sigma 54-holoenzyme form). To regulate the glnA promoter, NtrC binds to sites that have the properties of transcriptional enhancers: the sites will function far from the promoter and in an orientation-independent fashion. To investigate the mechanism of enhancer function, we have used electron microscopy to visualize the interactions of purified NtrC and RNA polymerase with their DNA binding sites and with each other. Under conditions that allow the formation of open complexes, about 30% of DNA molecules carry both RNA polymerase and NtrC bound to their specific sites. Of these, about 15% form looped structures in which NtrC and the RNA polymerase-promoter complex are in contact. The length of the looped DNA is that predicted from the spacing that was engineered between the enhancer and the glnA promoter (390 base pairs). As expected for activation intermediates, the looped structures disappear when RNA polymerase is allowed to transcribe the DNA. We conclude that the NtrC enhancer functions by means of a direct association between DNA-bound NtrC and RNA polymerase (DNA-looping model). Association of DNA-bound proteins appears to be the major mechanism by which different types of site-specific DNA transactions are localized and controlled.
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Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Porter
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Kustu
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Echols
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D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008854:Microscopy, Electron; D008957:Models, Genetic; D051922:PII Nitrogen Regulatory Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015183:Restriction Mapping; D015534:Trans-Activators; D014157:Transcription Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; D012321:DNA-Directed RNA Polymerases