Analysis of binding sites for the nitrogen-control transcription factor NtcA in the promoters of Synechococcus...

Dissociation constant (K(d)) was determined for the binding of the transcriptional regulator NtcA to a number of NtcA-activated promoters of the cyanobacterium Synechococcus sp. PCC 7942, as well as to mutant versions of those promoters altered in their NtcA-binding sites. K(d) values obtained ranged from 27 nM for the NtcA-binding site in the glnA promoter to ca. 1.4 microM for the site in the glnB promoter. Changes in the sequence signature of NtcA-binding sites (i.e. GTAN(8)TAC) either completely prevented binding of NtcA or drastically decreased its affinity for the promoter revealing that the GTN(10)AC subset is essential for binding. The importance of the flanking A/T-rich sequences and of a conserved CA dimer found in between the GTA/TAC triplets was also stressed. Thus, this structure seems to be optimal for the binding of NtcA. Comparison of K(d) values for NtcA binding to native promoters showed that differences in the sequence of their NtcA binding sites determine extensive differences in the affinity of the regulator for those sites.