Hacia el entendimiento del control de la expresión génica del regulón clanobacteriano del nitrógeno mediado por el factor de transcripción NtcA y la proteína reguladora PipX
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2018
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21-09-2018
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Abstract
NtcA is a cyanobacterial transcriptional regulator from the CRP family of transcription factors. It is considered as the global nitrogen regulator of cyanobacteria because it controls an extense regulon that includes many genes involved in nitrogen metabolism. NtcA is activated by 2-oxoglutarate (2-OG), an indicator of low nitrogen levels in the cell and it is coactivated by protein PipX, an important object of this study. At low 2-OG levels, PipX is sequestered by the PII signaling protein, which makes PipX inaccessible to activate NtcA. However, the binding of 2-OG to PII under nitrogen deprivation conditions releases PipX, rendering it accessible to co-activate NtcA. Structures of NtcA bound to 2-OG in the absence and in the presence of PipX had been previously determined in our laboratory; in this thesis we describe the structure of NtcA bound to its target DNA and also the structure of the NtcA-PipX-DNA complex. These structures clarifies the mechanism of NtcA specificity for its DNA box and also clearly show that PipX does not interact with DNA, but it stabilizes the active conformation of NtcA. Thus, the binding of PipX to NtcA displaces the balance between the multiple inactive forms of the transcription factor in favor of its DNA binding competent form. Here we present also the structure of three inactive forms of NtcA, without 2OG bound, demonstrating that the inactive conformation is not species specific, but that there are several inactive conformations for each species. In addition, in the PII-PipX complex, the C-terminal helix of PipX is extended, providing an opportunity of interaction with other possible targets, which could not occur in the NtcA-PipX complex, where this helix is always flexed. We have propitiated the determination of the PipX structure by NMR, proving that PipX when free, presents its C-terminal helix flexed. Recently, a putative gene regulator has been identified as a target of PipX in the PII-PipX-PlmA ternary complex. Since only PipX elements seem to interact with PlmA in this ternary complex, it seems that PII acts as an "opener" of the C-terminal helix of PipX, promoting the sequestration of PlmA in this complex. On the other hand, by using plasmon resonance technique, we have investigated the binding of NtcA and CRP proteins to promoters with canonical and non-canonical consensus sequences for these transcriptional regulators in the absence and in the presence of their respective effectors (2OG and cAMP respectively) and /or the PipX protein. These experiments were done in order to study what makes some genes of the NtcA regulon more or less sensitive to NtcA depending on the presence or absence of 2OG and PipX, and also to test the possibility of cross-activation betwen NtcA and CRP promoters, due to both transcription factors recognize similar DNA sequences. Our results show that NtcA is able bind to the CRP-dependent promoters but not the other way around. In addition to that, CRP is unable to bind to its own promoter in the absence of cAMP whereas NtcA does bind to its promoters in the absence of 2OG, albeit with less affinity than in his presence. We have also found that the effective affinity of NtcA for 2OG is highly influenced by the promoter and that PipX increases this affinity in an important way, interacting only with NtcA when 2OG is present, supporting the view that PipX stabilizes the active conformation of bound NtcA to 2OG.