Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking

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• 作者：Chen ; Shengfeng ; Gunasekera ; Angelo ; Zhang ; Xiaoping ; Kunkel ; Thomas A. ; Ebright ; Richard H. ; et. al.
• 关键词：catabolite activator protein (CAP) ; cAMP receptor protein (CRP) ; protein-DNA interaction ; indirect readout ; protein-induced DNA bending ; CAP ; catabolite activator protein
• 刊名：Journal of Molecular Biology
• 出版年：2001
• 出版时间：November 16, 2001
• 年：2001
• 卷：314
• 期：1
• 页码：75-82
• 全文大小：865 K

文摘

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of mages/glyphs/BQ1.GIF>40° and a twist angle of mages/glyphs/BQ1.GIF>20°, between positions 6 and 7 of the DNA half-site, 5′-A₁A₂A₃T₄G₅T₆G₇A₈T₉C₁₀T₁₁-3′ (“primary kink”). CAP recognizes the base-pair immediately 5′ to the primary-kink site, T:A₆, through an “indirect-readout” mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3′ to the primary-kink site, G:C₇, through a “direct-readout” mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C₇. Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A₆ to specificity for C:G₆, and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181→Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A₆. The Glu181→Asp substitution does not eliminate hydrogen-bond formation with G:C₇, and thus does not eliminate direct-readout-based specificity for G:C₇.