Lindemose, S.2; Nielsen, P. E.2; Valentin-Hansen, Poul3; Møllegaard, N. E.2
1 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU2 unknown3 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU
The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNN-NNTCACANTT through direct amino acid nucleobase interactions in the major groove of the two operator half-sites. Crystal structure analyses have revealed that the interaction results in two strong kinks at the TG/CA steps closest to the 6-base-pair spacer (N-6). This spacer exhibits high sequence variability among the more than 100 natural binding sites in the E. coli genome, but the exact role of the N-6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N-6 spacer region to demonstrate that CRP binding to the lacPI site may be enhanced up to 14-fold or abolished by varying the N-6 spacer sequences. Furthermore, on the basis of sequence analysis and uranyl (UO22+) probing data, we propose that the underlying mechanism relies on N-6 deformability.
A C S Chemical Biology, 2014, Vol 9, Issue 3, p. 752-760