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
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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DOI：
10.1006/jmbi.2001.5090
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发表时间：
2001-11-16
影响因子：
5.6
通讯作者：
Berman, HM
中科院分区：
文献类型：
--
作者：
Chen, SF;Gunasekera, A;Berman, HM
分解代谢激活蛋白（CAP）使CAP-DNA复合体中的DNA急剧弯曲，在DNA半位点的第6和第7个位置5'-A(1) a (2) a (3)T(4)G(5)T(6)G(7) a (8)T(9)C(10)T(11)-3'（“初级扭结”）之间引入一个DNA扭结，其滚动角类似于40度，扭转角类似于20度。CAP通过一种“间接读出”机制，直接识别到初级扭结位点T:A(6)的碱基对，该机制涉及到序列对初级扭结形成能量学的影响。通过在CAP的Glu181和G:C-7之间形成氢键的“直接读出”机制，CAP识别出与主扭结位点G:C-7直接相连的碱基对。在这里，我们报告了CAP的Glu181的羧酸侧链被Asp的一个亚甲基短羧酸侧链取代，改变了DNA半位点6位的DNA结合特异性，将T:A(6)的特异性改变为C:G(6)的特异性，我们报告了晶体学分析，定义了特异性变化的结构基础。Glu181 -> Asp取代消除了主要的扭结，从而消除了T:A的间接读出特异性(6)。Glu181 ->的Asp取代不会消除与G:C-7形成的氢键，因此不会消除基于直接读数的G:C-7特异性。(C) 2001学术出版社。
The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of similar to 40 degrees and a twist angle of similar to 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), 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:C-7, through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C-7. Here, we report that substitution of the carboxylate sidechain 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:A(6) to specificity for C:G(6), 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:A(6). The Glu181 --> Asp substitution does not eliminate hydrogen-bond formation with G:C-7, and thus does not eliminate direct-readout-based specificity for G:C-7. (C) 2001 Academic Press.