SPR analysis of promoter binding of <i>Synechocystis</i> PCC6803 transcription factors NtcA and CRP suggests cross‐talk and sheds light on regulation by effector molecules

Forcada-Nadal, Alicia; Forchhammer, Karl; Rubio, Vicente

doi:10.1016/j.febslet.2014.05.010

Cited by 29 publications

(28 citation statements)

References 37 publications

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“…Domain interactions in the activated form of NtcA are stabilized by the binding of PipX, which suggests that PipX drives the equilibrium toward the active conformation of NtcA (84,94). In agreement with this, biochemical studies revealed that PipX increases the affinity of NtcA for promoters and the effective affinity of NtcA for 2-OG (88). Modeling of the PipX-NtcA complex on DNA suggests PipX is shown as a surface structure, and 2-OG is shown as blue and red spheres.…”

Section: The Ntca Transcriptional Regulator In Cyanobacteriamentioning

confidence: 62%

“…5). This may explain why binding of 2-OG to NtcA results in a relatively small enhancement in the affinity for DNA, compared with a large change in affinity brought about by the binding of cAMP to Crp (85,88). Structural characterization of the 2-OG binding site in NtcA has been valuable for designing chemical derivatives of 2-OG that mimic its signaling function in vivo (89).…”

Section: The Ntca Transcriptional Regulator In Cyanobacteriamentioning

confidence: 99%

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The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite

Huergo

Dixon

2015

Microbiol Mol Biol Rev

241

221

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SUMMARYThe metabolite 2-oxoglutarate (also known as α-ketoglutarate, 2-ketoglutaric acid, or oxoglutaric acid) lies at the intersection between the carbon and nitrogen metabolic pathways. This compound is a key intermediate of one of the most fundamental biochemical pathways in carbon metabolism, the tricarboxylic acid (TCA) cycle. In addition, 2-oxoglutarate also acts as the major carbon skeleton for nitrogen-assimilatory reactions. Experimental data support the conclusion that intracellular levels of 2-oxoglutarate fluctuate according to nitrogen and carbon availability. This review summarizes how nature has capitalized on the ability of 2-oxoglutarate to reflect cellular nutritional status through evolution of a variety of 2-oxoglutarate-sensing regulatory proteins. The number of metabolic pathways known to be regulated by 2-oxoglutarate levels has increased significantly in recent years. The signaling properties of 2-oxoglutarate are highlighted by the fact that this metabolite regulates the synthesis of the well-established master signaling molecule, cyclic AMP (cAMP), inEscherichia coli.

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Section: The Ntca Transcriptional Regulator In Cyanobacteriamentioning

confidence: 62%

Section: The Ntca Transcriptional Regulator In Cyanobacteriamentioning

confidence: 99%

The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite

Huergo

Dixon

2015

Microbiol Mol Biol Rev

241

221

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“…The subdivision of Cluster I transcripts into two sub-clusters with expression patterns suggestive of PipX and PipY having independent (IA) or additive (IB) effects, respectively, can be rationalized on the bases of the affinity of NtcA for its different binding sites (Espinosa et al, 2007; Forcada-Nadal et al, 2014). NtcA targets from Cluster IB would provide weaker NtcA binding and thus greater dependency on both PipX and PipY.…”

Section: Discussionmentioning

confidence: 99%

“…The PipX protein can form alternative complexes with NtcA and PII and these interactions are, respectively, stimulated and inhibited by 2-OG, providing a mechanistic link between PII signaling and NtcA-regulated gene expression (Espinosa et al, 2006). PipX binds to either, 2-OG-bound NtcA to stimulate DNA binding and transcriptional activity, or to 2-OG-free PII to form PII-PipX complexes (Tanigawa et al, 2002; Vazquez-Bermudez et al, 2002; Llacer et al, 2010; Zhao et al, 2010; Forcada-Nadal et al, 2014), which in turn can bind to the transcriptional regulator PlmA (Labella et al, 2016). While PII is found in all three domains of life as integrator of signals of the nitrogen and carbon balance, PipX, NtcA, and PlmA are all restricted to cyanobacteria.…”

Section: Introductionmentioning

confidence: 99%

PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

et al. 2017

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Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essential PLP-holoenzymes or synthetic lethality with cysK. In addition, the related phenotypes of pipY and pipX mutants are consistent with genetic interactions in the contexts of survival to PLP-targeting antibiotics and transcriptional regulation. We also showed that PipY overexpression increased the length of S. elongatus cells. Taken together, our results support a universal regulatory role for COG0325 proteins, paving the way to a better understanding of these proteins and of their connections with other biological processes.

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“…PCC 7120 (hereafter S. elongatus and Anabaena , respectively) showed that activation of NtcA-dependent genes is enhanced by PipX binding to NtcA (Espinosa et al, 2006, 2007; Valladares et al, 2011; Forcada-Nadal et al, 2014). Under nitrogen limitation, 2-OG stimulates NtcA binding to target sites (Vázquez-Bermúdez et al, 2002), activating transcription (Tanigawa et al, 2002) and favoring complex formation between NtcA and PipX (Espinosa et al, 2006) (Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

Expanding the Cyanobacterial Nitrogen Regulatory Network: The GntR-Like Regulator PlmA Interacts with the PII-PipX Complex

et al. 2016

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Cyanobacteria, phototrophic organisms that perform oxygenic photosynthesis, perceive nitrogen status by sensing 2-oxoglutarate levels. PII, a widespread signaling protein, senses and transduces nitrogen and energy status to target proteins, regulating metabolism and gene expression. In cyanobacteria, under conditions of low 2-oxoglutarate, PII forms complexes with the enzyme N-acetyl glutamate kinase, increasing arginine biosynthesis, and with PII-interacting protein X (PipX), making PipX unavailable for binding and co-activation of the nitrogen regulator NtcA. Both the PII-PipX complex structure and in vivo functional data suggested that this complex, as such, could have regulatory functions in addition to PipX sequestration. To investigate this possibility we performed yeast three-hybrid screening of genomic libraries from Synechococcus elongatus PCC7942, searching for proteins interacting simultaneously with PII and PipX. The only prey clone found in the search expressed PlmA, a member of the GntR family of transcriptional regulators proven here by gel filtration to be homodimeric. Interactions analyses further confirmed the simultaneous requirement of PII and PipX, and showed that the PlmA contacts involve PipX elements exposed in the PII-PipX complex, specifically the C-terminal helices and one residue of the tudor-like body. In contrast, PII appears not to interact directly with PlmA, possibly being needed indirectly, to induce an extended conformation of the C-terminal helices of PipX and for modulating the surface polarity at the PII-PipX boundary, two elements that appear crucial for PlmA binding. Attempts to inactive plmA confirmed that this gene is essential in S. elongatus. Western blot assays revealed that S. elongatus PlmA, irrespective of the nitrogen regime, is a relatively abundant transcriptional regulator, suggesting the existence of a large PlmA regulon. In silico studies showed that PlmA is universally and exclusively found in cyanobacteria. Based on interaction data, on the relative amounts of the proteins involved in PII-PipX-PlmA complexes, determined in western assays, and on the restrictions imposed by the symmetries of trimeric PII and dimeric PlmA molecules, a structural and regulatory model for PlmA function is discussed in the context of the cyanobacterial nitrogen interaction network.

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

Cited by 29 publications

References 37 publications

The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite

The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite

PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

Expanding the Cyanobacterial Nitrogen Regulatory Network: The GntR-Like Regulator PlmA Interacts with the PII-PipX Complex

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