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

Finney, Angela H.2014-03-142014-03-142000-12-15etd-12192000-174959http://hdl.handle.net/10919/36285Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, Vibrio fischeri. In V. fischeri, the luminescence or lux 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 lux 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 aCTD of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effect of seventy alanine substitution variants of the a subunit was determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by in vitro assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxRDN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRDN, and fourteen residues in the aCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxRDN-dependent activation in vivo and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type aCTD is capable of interacting with the lux DNA fragment tested, all five of the variant forms of the aCTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that aCTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the lux operon during quorum sensing.enIn CopyrightRNA polymerasetranscriptional activationDNA bindingLuxRquorum sensingVibrio fischerialpha subunitluminescenceRole of the C-terminal domain of the a subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensingThesishttp://scholar.lib.vt.edu/theses/available/etd-12192000-174959/