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Promoter architecture and transcriptional regulation by the NAC protein of Klebsiella aerogenes.

dc.contributor.authorPomposiello, Pablo Jose
dc.contributor.advisorBender, Robert A.
dc.date.accessioned2016-08-30T17:29:42Z
dc.date.available2016-08-30T17:29:42Z
dc.date.issued1997
dc.identifier.urihttp://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9732162
dc.identifier.urihttps://hdl.handle.net/2027.42/130564
dc.description.abstractThe NAC protein of Klebsiella aerogenes is involved in a regulatory cascade that allows the cell to adapt to changes in the quality and quantity of available nitrogen-containing compounds in the medium. NAC is a DNA-binding protein that activates transcription of genes whose products degrade nitrogen-containing compounds, and represses transcription of genes whose products use nitrogen for biosynthesis. Previous work showed that transcriptional activation by NAC requires protein binding to regions of DNA that contain the partially palindromic site ATA-N9-TAT centered at position $-$64. The NAC binding site from the histidine utilization promoter (hutUp) was sufficient to confer regulation by NAC to three semi-synthetic promoters. The displacement of the NAC site relative to these promoters showed that NAC activates transcription from positions $-$64, $-$53 and $-$43, but fails to activate from positions $-$74, $-$69, $-$59 and $-$49. Random and site-directed mutagenesis of the NAC binding site from hutUp revealed that substitution of the conserved nucleotides in the palindromic ends of the binding site, ATA and TAT, reduced protein binding and transcriptional activation. Mutations in between these ends stimulated or decreased protein binding and transcriptional activation, while mutations outside these ends had only marginal effects on transcriptional activation. The replacement of the NAC binding site from hutUp in a semi-synthetic promoter by the NAC binding site from the glutamate dehydrogenase gene resulted in the failure of NAC to activate transcription, despite similar binding affinity and protein-DNA complex structure. In addition, two mutations in the promoter-proximal half site of the NAC binding increased protein binding affinity, but decreased activation. These results suggest two roles for the NAC binding site: the binding of the protein at the right distance and face of the DNA helix, and the presentation of the protein in a functional configuration.
dc.format.extent127 p.
dc.languageEnglish
dc.language.isoEN
dc.subjectAerogenes
dc.subjectArchitecture
dc.subjectKlebsiella
dc.subjectNac
dc.subjectPromoter
dc.subjectProtein
dc.subjectRegulati
dc.subjectRegulation
dc.subjectTranscriptional
dc.titlePromoter architecture and transcriptional regulation by the NAC protein of Klebsiella aerogenes.
dc.typeThesis
dc.description.thesisdegreenamePhDen_US
dc.description.thesisdegreedisciplineBiological Sciences
dc.description.thesisdegreedisciplineGenetics
dc.description.thesisdegreedisciplineMicrobiology
dc.description.thesisdegreedisciplineMolecular biology
dc.description.thesisdegreegrantorUniversity of Michigan, Horace H. Rackham School of Graduate Studies
dc.description.bitstreamurlhttp://deepblue.lib.umich.edu/bitstream/2027.42/130564/2/9732162.pdf
dc.owningcollnameDissertations and Theses (Ph.D. and Master's)


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