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, glnN (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 PII-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 (Kd≈85 nM) than to the PipX-sequestering PII protein. NtcA appears to require more strongly PipX and 2-oxoglutarate (2OG) for estimulating gene expression at promoters having "imperfect" NtcA binding sites.
Instituto de Biomedicina de Valencia of the CSIC, Valencia, Spain.
Alicia
A
Forcada-Nadal
Interfaculty Institute for Microbiology and Infection Medicine, Division Organismic Interactions, University of Tübingen, Germany. Electronic address: karl.forchhammer@uni-tuebingen.de.
Karl
K
Forchhammer
Instituto de Biomedicina de Valencia of the CSIC, Valencia, Spain; Group 739 of the Centro de Investigación Biomédica en Red sobre Enfermedades Raras (CIBERER) del Instituto de Salud Carlos III, Spain. Electronic address: rubio@ibv.csic.es.
Vicente
V
Rubio
D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D001483:Base Sequence; D000242:Cyclic AMP / Q000737:chemistry; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000502:physiology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007656:Ketoglutaric Acids / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D046939:Synechocystis / Q000235:genetics*
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D007656:Ketoglutaric Acids; D000242:Cyclic AMP
2-Oxoglutarate; Catabolite gene activator protein; Cyanobacteria; Nitrogen metabolism; PipX protein; Protein–protein interaction