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Author Patterson-Fortin, Laura Margaret
Title LexA-mediated repression of the cyanobacterial RNA helicase, crhR
book jacket
Descript 188 p
Note Source: Dissertation Abstracts International, Volume: 70-02, Section: B, page: 0821
Thesis (Ph.D.)--University of Alberta (Canada), 2008
Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions which elicit reduction of the photosynthetic electron transport chain. Transcriptional regulation of crhR expression was investigated in this thesis. DNA affinity chromatography and mass spectrometry identified that the Synechocystis sp. strain PCC 6803 LexA orthologue binds the crhR gene. Recombinant LexA (rLexA) interacts specifically with both the crhR and lexA genes identifying both as LexA targets. Transcript analysis was initially used to investigate the LexA/crhR regulatory relationship and indicated that lexA and crhR are divergently expressed under the redox conditions examined suggesting LexA is a negative regulator of crhR expression. In vivo and in vitro analysis confirmed LexA repression of crhR expression. rLexA decreases expression in a linear manner in an in vitro transcriptional/translation while in vivo reduction of lexA levels in a lexA heteroploid mutant correlated with increased accumulation of the crhR transcript under oxidizing conditions. Transcript analysis also demonstrated that expression of the crhR, recA and lexA genes in Synechocystis is not inducible by DNA damage
A combination of DNaseI footprinting, site directed mutagenesis and electrophoretic mobility shift assays identified that the LexA-orthologue binds to a CTA-N 9-CTA direct repeat conserved within the open reading frame and the 5' untranslated region of the crhR and lexA genes, respectively. Furthermore, gel exclusion chromatography and an electrophoretic mobility shift assay-based method were used to demonstrate that rLexA exists as a monomer in solution and as a dimer when bound to DNA
Insertional inactivation of crhR and lexA was used to further investigate the physiological roles of both proteins in the cell. crhR is a non-essential gene. CrhR is required at low temperature as demonstrated by the absence of growth at 20°C. In contrast, lexA is an essential gene, evidenced by the continual maintenance of wild type copies of the gene after several generations on selective medium
The potential significance of a LexA-orthologue in the regulation of redox-responsive gene expression and consequently, the implications of this novel role performed by LexA in Synechocystis are discussed
School code: 0351
Host Item Dissertation Abstracts International 70-02B
Subject Biology, Molecular
Biology, Microbiology
0307
0410
Alt Author University of Alberta (Canada)
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