SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross-talk and sheds light on regulation by effector molecules

Surface plasmon resonance monitoring of the binding of transcription factors cAMP receptor protein (CRP) and nitrogen control factor of cyanobacteria (NtcA) from Synechocystis sp. PCC6803 to promoter fragments of glnA, gInN (NtcA regulon) and cccS (CRP regulon), revealed exclusive CRP binding to cccS, whereas NtcA was bound to all three promoters with different affinities, which were strongly increased by the NtcA activator 2-oxoglutarate. Effective NtcA affinity for 2-oxoglutarate varied with the promoter. High-affinity promoters and the NtcA-coactivating protein P11-interacting protein X (PipX) increased NtcA affinity towards 2-oxoglutarate, suggesting PipX-stabilization of the 2-oxoglutarate-bound NtcA conformation. PipX binding to NtcA required 2-oxoglutarate and was much tighter (K-d approximate to 85 nM) than to the PipX-sequestering PH protein. NtcA appears to require more strongly PipX and 2-oxoglutarate (20G) for estimulating gene expression at promoters having imperfect NtcA binding sites. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.