In Salmonella typhimurium and Escherichia coli cysteine biosynthesis is controlled by a combination of feedback inhibition of serine transacetylase and a system of positive gene regulation termed the cysteine regulon, in which the CysB protein positively regulates expression of genes of the biosynthetic pathway. CysB protein interacts with the promoter region of these genes, and in the presence of inducer, which can be either O- acetylserine or N-acetylserine, stimulates formation of transcription initiation complexes. Sulfide acts as an anti-inducer, which competes with acetylserine. CysB protein also autoregulates its own synthesis by binding to the cysB promoter region, where it acts as a repressor rather than a transcriptional activator. Whereas binding to positively regulated promoters is stimulated by acetylserine, binding to the cysB promoter is inhibited. In order to define more precisely the attributes of the different kinds of DNA sequences that bind CysB protein, the promoter regions from cys genes will be sequenced and compared by means of footprinting, in vitro transcription studies and site-directed mutagenesis. Interactions of CysB protein with DNA binding sites, acetylserine and the anti-inducer sulfide will be studied by physicochemical and genetic means. Effects of acetylserine and sulfide on CysB protein subunit structure will be determined by sedimentation velocity and equilibrium studies. the stoichiometry of protein binding to different promoters will be analyzed with radiolabelled protein and DNA. Site-directed mutagenesis will be used to generate cysB alleles that are resistant to sulfide and no longer require acetylserine to promoters will be analyzed in vitro. Protein domains interacting with DNA and acetylserine will be identified through the creation of analysis of chimeric proteins containing sequences from CysB protein and from closely related regulatory proteins, which are thought to have a common mechanism of action even though they respond to different inducers. The enzyme sulfite reductase catalyzes the reduction of sulfite to sulfide and consists of two different portions, a flavoprotein and a hemoprotein, which contains an iron-sulfide cluster and siroheme. Site-directed mutagenesis of cysl, the gene encoding the hemoprotein, will be carried out in an attempt to identify the amino acid residue that transfers electrons from the iron-sulfide cluster to siroheme. Mutant proteins will be purified and studied by enzymatic and spectroscopic methods. Similar studies will be carried out with the enzyme O-acetylserine (thiol) lyase, which catalyzes the synthesis of cysteine from O-acetylserine and sulfide.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Biochemistry Study Section (BIO)
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Duke University
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