Interaction ofBacillus subtilisCodY with GTP

Many of the adaptive mechanisms that allow Bacillus subtilis to adjust to changes in nutrient availability are controlled by CodY. Binding of CodY to its target genes is stimulated by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon nutrient limitation, intracellular pools of these effectors are depleted and CodY can no longer repress genes required for adaptation. In vitro studies reported here explored in more detail the interaction of CodY with GTP. DNase I footprinting experiments indicated that CodY has an affinity for GTP in the millimolar range. Further, CodY was shown to interact specifically with GTP and dGTP; no other naturally occurring nucleotides that were tested, including ppGpp and pppGpp, resulted in DNA protection. Two nonhydrolyzable analogs of GTP were fully able to activate CodY binding to target DNA, demonstrating that GTP hydrolysis is not necessary for CodY-dependent regulation. GTP and the BCAAs were shown to act additively to increase the affinity of CodY for DNA; increased protection was observed in DNase I footprinting experiments when both effectors were present, compared to either effector alone, and in in vitro transcription reactions, transcriptional repression by CodY was stronger in the presence of both GTP and BCAAs than of BCAAs alone. Thus, interaction of CodY with GTP is specific and results in increased affinity for its target genes. This increase in affinity is independent of GTP hydrolysis and is augmented in the presence of BCAAs.