Protein-DNA interactions in the regulation of lipid biosynthesis in Bacillus subtilis

GEORGINA REH; GUSTAVO E. SCHUJMAN; DIEGO DE MENDOZA

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigadores en Bioquímica (SAIB); 2007

SAIB

Protein-DNA interactions in the regulation of lipid biosynthesis in Bacillus subtilis Reh G, Schujman GE, de Mendoza IBR - CONICET, Facultad Cs Bioq y Farm, UNR, Suipacha 531, 2000 Rosario, Argentina. E-mail: reh@ibr.com.ar FapR is a global transcriptional repressor that controls the expression of many genes involved in the biosynthesis of lipids (the fap regulon) in Bacillus subtilis. FapR has highly conserved homologues in many Gram-positive bacteria, including several human pathogens. As well, in all these organisms the consensus binding sequence of FapR is largely invariant in the putative promoter regions of the fapR gene, indicating that the regulation mechanism is conserved. FapR belongs to a new class of bacterial repressors in which malonyl-CoAoperates as the direct and specific inducer of FapR-regulated promoters. Despite genetic and biochemical studies, specific details of the interaction between FapR and its operator sequences remain unknown. To obtain a high resolution profile of the contacts between FapR and its target DNA we resorted to hydroxyl radical footprinting analysis and gel shift assays. For each promoter analyzed, four protected regions spaced from 6 to 9 nucleotides were detected. Also, in each case, two different complexes were observed in gel shifts assays. To confirm the recognition motifs, these assays were repeated with promoter variants carrying specific mutations. Our results strongly suggest that FapR binds to its operators as a tetramer or two dimers in contact with the DNA backbone at the same face of the DNA helix.