The mechanism of gene regulation used by Gram-negative bacteria during quorum sensing is well understood in the bioluminescent marine bacterium Vibrio fischeri. The cell-density dependent activation of the luminescence (lux) genes of V. fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl) homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. LuxR, a 250 amino acid polypeptide, binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site. During transcriptional activation of the lux operon, LuxR is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli sigma 70 subunit of RNAP in LuxR-dependent transcriptional activation of the luxI promoter has been investigated. Rich in basic amino acids, this conserved portion of sigma 70 is likely to be surface-exposed and available to interact with transcription factors bound near the -35 element. The effect of 16 single and 2 triple alanine substitution variants of sigma 70 between amino acid residues 590 and 613, was determined in vivo by measuring the rate of transcription from a luxI-lacZ translational fusion via b-galactosidase assays in recombinant E. coli. In vitro work was performed with LuxRDN, the autoinducer-independent C-terminal domain (amino acids 157 to 250) of LuxR because purified, full length LuxR is unavailable. Single-round transcription assays were performed in the presence of LuxRDN and 19 variant RNAPs, one of which contained a C-terminally truncated sigma 70 subunit devoid of region 4. Results indicate that region 4 is essential for LuxRDN-dependent luxI transcription with two specific amino acid residues, E591 and K597, having negative effects on the rate of LuxRDN-dependent luxI transcription in vivo and in vitro. None of the residues tested were identified as having any effect on LuxR-dependent luxI transcription in vivo. These findings suggest that region 4.2 is most likely to be in close proximity to LuxR when bound to the luxI promoter. However, unlike the situation found for other ambidextrous activators, no single residue within region 4.2 of sigma 70 may be critical by itself for LuxR-dependent during transcriptional activation. / Master of Science
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/31680 |
| Date | 18 April 2002 |
| Creators | Johnson, Deborah Cumaraswamy |
| Contributors | Biology, Stevens, Ann M., Rutherford, Charles L., Popham, David L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | dcjthesis.pdf |
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