Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system.
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.
Lindemose
Søren
S
Department of Cellular and Molecular Medicine, Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Nielsen
Peter Eigil
PE
Møllegaard
Niels Erik
NE
D015075:2-Aminopurine / Q000031:analogs & derivatives* / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D015723:Gene Library; D007288:Inosine / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D011953:Receptors, Cyclic AMP / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
D004268:DNA-Binding Proteins; D003854:Deoxyribonucleotides; D029968:Escherichia coli Proteins; D011953:Receptors, Cyclic AMP; C047947:cyclic AMP receptor protein, E coli; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D007288:Inosine; D004247:DNA