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

TY - JOUR

T1 - Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system.

AU - Lindemose, Søren

AU - Nielsen, Peter E.

AU - Møllegaard, Niels Erik

N1 - Keywords: 2-Aminopurine; Base Sequence; Binding Sites; Consensus Sequence; DNA; DNA Footprinting; DNA-Binding Proteins; Deoxyribonucleotides; Electrophoretic Mobility Shift Assay; Escherichia coli Proteins; Gene Library; Inosine; Polymerase Chain Reaction; Protein Binding; Receptors, Cyclic AMP; Sequence Alignment

PY - 2008

Y1 - 2008

N2 - 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.

AB - 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.

U2 - 10.1093/nar/gkn452

DO - 10.1093/nar/gkn452

M3 - Journal article

C2 - 18653536

SN - 0305-1048

VL - 36

SP - 4797

EP - 4807

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 14

ER -