Role of the C-terminal domain of the a subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing

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dc.contributor.authorFinney, Angela H.en
dc.contributor.committeechairStevens, Ann M.en
dc.contributor.committeememberPopham, David L.en
dc.contributor.committeememberRutherford, Charles L.en
dc.contributor.departmentBiology (Microbiology)en
dc.date.accessioned2014-03-14T20:50:21Zen
dc.date.adate2000-12-20en
dc.date.available2014-03-14T20:50:21Zen
dc.date.issued2000-12-15en
dc.date.rdate2001-12-20en
dc.date.sdate2000-12-19en
dc.description.abstractQuorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, <i>Vibrio fischeri</i>. In <i>V. fischeri</i>, the luminescence or <i>lux</i> genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the <i>lux</i> operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the <font face = "symbol">a</font>CTD of RNAP in LuxR-dependent transcriptional activation of the <i>lux</i> operon promoter has been investigated. The effect of seventy alanine substitution variants of the <font face = "symbol">a</font> subunit was determined <i>in vivo</i> by measuring the rate of transcription of the <i>lux</i> operon via luciferase assays in recombinant <i>Escherichia coli</i>. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by <i>in vitro</i> assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxR<font face = "symbol">D</font>N, was used for <i>in vitro</i> studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxR<font face = "symbol">D</font>N, and fourteen residues in the <font face = "symbol">a</font>CTD were identified as having negative effects on the rate of transcription from the <i>lux</i> operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxR<font face = "symbol">D</font>N-dependent activation <i>in vivo</i> and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type <font face = "symbol">a</font>CTD is capable of interacting with the <i>lux</i> DNA fragment tested, all five of the variant forms of the <font face = "symbol">a</font>CTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that <font face = "symbol">a</font>CTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the <i>lux</i> operon during quorum sensing.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-12192000-174959en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-12192000-174959/en
dc.identifier.urihttp://hdl.handle.net/10919/36285en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartFINALetd12_20.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectRNA polymeraseen
dc.subjecttranscriptional activationen
dc.subjectDNA bindingen
dc.subjectLuxRen
dc.subjectquorum sensingen
dc.subjectVibrio fischerien
dc.subjectalpha subuniten
dc.subjectluminescenceen
dc.titleRole of the C-terminal domain of the <font face = "symbol">a</font> subunit of RNA polymerase in transcriptional activation of the <i>lux</i> operon during quorum sensingen
dc.typeThesisen
thesis.degree.disciplineBiology (Microbiology)en
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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