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Bacteria belonging to the Gram positive genus Streptomyces produce many secondary metabolite natural products of pharmaceutical importance and undergo a complex program of morphological differentiation. The expression of genes involved in these processes is often regulated by γ-butyrolactone (GBL) interactions with cognate GBL receptors. GBL receptors regulate gene expression by binding specific AT-rich DNA sequences known as autoregulatory elements (ARE) in response to available intracellular GBL pools. We previously identified two S. acidiscabies GBL receptor genes, sabR and sabS, flanking a gene encoding a GBL synthase homolog sabA. Genetic and biochemical studies revealed that sabR negatively regulates sabS expression by binding an ARE located in the 5’ region of sabS, while sabS mutants overproduce the type II aromatic polyketide WS5995B and exhibit a conditional impairment in morphological differentiation. In order to further understand the role of GBL receptors in regulating cellular behavior, we have applied synthetic and genomic SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methods to identify AREs for recombinant SabR and SabS. Following variable rounds of iterative SELEX, DNA substrates bound to the proteins were cloned and sequenced. Sequence analysis of cloned products revealed DNA sequences that resemble AT-rich ARE sequences. This method can now be used to identify target genes regulated by these GBL receptors and compare the properties of binding interactions of GBL receptors with physiological and synthetic DNA substrates.
Spaulding, Caitin Nicole, "SELEX-Based Identification of Synthetic Binding Sites for Streptomyces acidiscabies 84.104 γ-Butyrolactone Receptors: SabS and SabR." (2011). Biology Honors Theses. 8.
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