Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: Recognition of pyrimidine-purine and purine-purine steps

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Abstract

The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of ∼40° and a twist angle of ∼20°, between positions 6 and 7 of the DNA half-site, 5′-A1A2A3T4G5T 6G7A8T9C10T 11-3′ ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5′ to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of ∼42°, twist angles of ∼16°), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of ∼20°, twist angles of ∼17°). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, ∼46° per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, ∼28° per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.

Original languageEnglish (US)
Pages (from-to)173-183
Number of pages11
JournalJournal of molecular biology
Volume357
Issue number1
DOIs
Publication statusPublished - Mar 17 2006

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All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • Catabolite activator protein (CAP)
  • Indirect readout
  • Protein-DNA interaction
  • Protein-induced DNA bending
  • cAMP receptor protein (CRP)

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