A Novel Indirect Sequence Readout Component in the E. coli Cyclic AMP Receptor Protein Operator

作者：Lindemose Soren; Nielsen Peter Eigil; Valentin Hansen Poul; Mollegaardt Niels Erik^*

来源：ACS Chemical Biology, 2014, 9(3): 752-760.

DOI：10.1021/cb4008309

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.

• 出版日期2014-3