Lindemose Søren, Nielsen Peter Eigil, Møllegaard Niels Erik
Department of Cellular and Molecular Medicine, Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Nucleic Acids Res. 2008 Aug;36(14):4797-807. doi: 10.1093/nar/gkn452. Epub 2008 Jul 24.
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 TGTGAN(6)TCACA (i.e. TITIDN(6)TCDCD). 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.
大肠杆菌环磷酸腺苷受体蛋白（CRP）与DNA的相互作用代表了一种典型的双重识别机制，兼具直接读出和间接读出。我们通过体外筛选一个随机的DNA结合位点文库来剖析CRP对DNA识别的直接和间接成分，该文库分别用次黄嘌呤（I）和2,6 -二氨基嘌呤（D）取代了鸟嘌呤和腺嘌呤。相应地，由于鸟嘌呤（现为I）的2 -氨基“转移”到腺嘌呤（现为D），DNA螺旋小沟的结构发生改变，而大沟在功能上保持完整。大多数筛选出的位点包含天然共有序列TGTGAN(6)TCACA（即TITIDN(6)TCDCD）。因此，共有序列的直接读出与小沟构象无关。所以，已知在共有序列的TG/CA碱基对步（主要扭结位点）发生的间接读出不受I - D替换的影响。相反，侧翼区域被选择为富含I/C的序列（大多为I链），而不是已知能强烈增强CRP结合的富含A/T的序列，从而表明通过I链的（各向异性）柔韧性，该区域几乎完全是对螺旋结构/柔韧性的间接读出。
