Probing activation of the prokaryotic arginine transcriptional regulator using chimeric proteins.

摘要: Abstract The major transcription factors controlling arginine metabolism in Escherichia coli and Bacillus subtilis, ArgR AhrC, respectively, are homologous multimeric proteins that form l -arginine-dependent DNA-binding complexes capable of repressing the biosynthetic genes (both), activating catabolic (AhrC only) or facilitating plasmid dimer resolution (both). Multimerisation -arginine binding associated with C-terminal 70-80 residues; N-terminal regions contain a winged helix-turn-helix domain. We have constructed chimeric which sequences for N domains been swapped. resultant their corresponding native analysed ability to multimerise bind DNA operator sites an fashion. Gel filtration equilibrium sedimentation analysis consistent formation hexamers by all four presence at high protein concentrations (>100 nM monomer). hexamer coefficients suggest there is reduction molecular volume upon L -arginine, conformational change accompanying allosteric activation DNA-binding. In absence lower concentrations, clearly rapid smaller subunits, whose dominant species appear be based on trimers, as expected from crystal structure fragment, exception ArgR-C chimera, apparently dissociates into dimers, suggesting intact may significant dimeric interaction. hexamer-trimer K d micromolar range, trimers principal vivo concentrations. has probed gel retardation DNase I footprinting using three types naturally occurring operators: encompassing two 18 bp ARG boxes separated 2 bp; containing such third box distance 100 bp downstream, i.e. within structural gene; finally contains single site. data show operators From apparent affinities chimeras each target site, no obvious sequence-specificity domains; rather can interpreted terms differential activation, including -arginine. Remarkably, “anti-competition” excess, specific fragments retardation. This appears due assembly activated protein, probably hexamers, DNA. discussed current models mode action both proteins.