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Nucleic Acids Research Advance Access published online on July 24, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn452
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Structural Biology

Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system

Søren Lindemose, Peter Eigil Nielsen and Niels Erik Møllegaard*

Department of Cellular and Molecular Medicine, Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark

*To whom correspondence should be addressed. Tel: +45 35327778; Fax: +45 35326042; Email: nem{at}imbg.ku.dk

Received March 31, 2008. Revised June 28, 2008. Accepted June 30, 2008.

The DNA interaction of the Escherichia coli cyclic AMP receptor protein (CRP) represents a typical example of a dual recognition mechanism exhibiting both direct and indirect readout. We have dissected the direct and indirect components of DNA recognition by CRP employing in vitro selection of a random library of DNA-binding sites containing inosine (I) and 2,6-diaminopurine (D) instead of guanine and adenine, respectively. Accordingly, the DNA helix minor groove is structurally altered due to the ‘transfer’ of the 2-amino group of guanine (now I) to adenine (now D), whereas the major groove is functionally intact. The majority of the selected sites contain the natural consensus sequence TGTGAN6TCACA (i.e. TITIDN6TCDCD). Thus, direct readout of the consensus sequence is independent of minor groove conformation. Consequently, the indirect readout known to occur in the TG/CA base pair step (primary kink site) in the consensus sequence is not affected by I–D substitutions. In contrast, the flanking regions are selected as I/C rich sequences (mostly I-tracts) instead of A/T rich sequences which are known to strongly increase CRP binding, thereby demonstrating almost exclusive indirect readout of helix structure/flexibility in this region through (anisotropic) flexibility of I-tracts.


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