Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library

A recursive in vitro selection among random DNA sequences was used for anal ysis of the cyanobacterial transcription factor NtcA-binding motifs. An eig ht-base palindromic sequence, TGTA-(N-8)-TACA, was found to be the optimal NtcA-binding sequence. The more divergent the binding sequences, compared t o this consensus sequence, the lower the NtcA affinity. The second and thir d bases in each four-nucleotide half of the consensus sequence were crucial for NtcA binding, and they were in general highly conserved. The most freq uently occurring sequence in the middle weakly conserved region was similar to that of the NtcA-binding motif of the Anabaena sp. strain PCC 7120 glnA gene, previously known to have high affinity for NtcA. This indicates that the middle sequences were selected for high NtcA affinity. Analysis of nat ural NtcA-binding motifs showed that these could be classified into two gro ups based on differences in recognition consensus sequences. It is suggeste d that NtcA naturally recognizes different DNA-binding motifs, or has diffe rential affinities to these sequences under different physiological conditi ons. (C) 2000 Academic Press.