An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage lambda cI repressor.
An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage lambda cI repressor.
复制标题
亚基之间的芳香堆积相互作用有助于介导 DNA 序列特异性：噬菌体 lambda cI 阻遏物对操作位点的辨别。
DOI：
10.1006/jmbi.1996.0869
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发表时间：
1997
影响因子：
5.6
通讯作者：
Senear,DF
中科院分区：
文献类型：
--
作者：
Huang,YT;Rusinova,E;Ross,JB;Senear,DF
调节蛋白的序列特异性 DNA 结合为转录起始的调节提供了基础。在理解单个蛋白质亚基的序列特异性识别方面已经取得了很大进展。需要理解的另一个控制水平是由于寡聚调节蛋白亚基之间的偶联。噬菌体 λ cI 阻遏蛋白就是一个例子，它是一种二聚体蛋白，可调节噬菌体从溶原性到裂解性的基因开关。两个层面的特异性对该法规至关重要。首先，像所有转录调节因子一样，二聚体将操纵基因与非特异性 DNA 区分开来。通过识别螺旋直接读出 DNA 序列被认为是众所周知的机制。然而，OR1、OR2 和 OR3 的不同亲和力对于开关同样重要，因为它介导不同启动子的相反调节。第二级的位点特异性尚不清楚。阻遏蛋白和不同操纵子的构象适应似乎很重要。为了评估亚基-亚基相互作用如何参与这一过程，我们研究了二聚体界面中对称相关螺旋 5 之间接触处的氨基酸取代对二聚体稳定性和操纵子结合的影响。 Tyr88 的取代会改变二聚体稳定性并极大地扰乱差异操纵子亲和力，但通常不会影响操纵子与非操纵子的特异性。这些效应的模式表明，每个亚基中对称相关的 Tyr88（二聚体界面中的一个基团，但远离 DNA 结合界面的基团）之间面对面芳香堆积相互作用的几何形状在操作者辨别中发挥着关键作用。似乎涉及亚基三级结构的构象变化。相比之下，I84S 替换的显着效果是大大降低了所有三个运算符的亲和力。据推测，二聚体界面的堆积变化导致了四级结构变化，使两个螺旋-转角-螺旋基序与连续的 DNA 大沟不对齐。
Sequence specific DNA binding by regulatory proteins provides the basis for regulation of initiation of transcription. A great deal of progress has been made toward understanding sequence specific recognition by individual protein subunits. An additional level of control that needs to be understood is that due to coupling between the subunits of oligomeric regulatory proteins. An example is the bacteriophage λ cI repressor, a dimeric protein that regulates the lysogenic to lytic genetic switch of the phage. Two levels of specificity are critical to this regulation. First, like all transcriptional regulators, dimers distinguish operator from non-specific DNA. Direct readout of the DNA sequence by the recognition helix is considered the well understood mechanism for this. However, differential affinity for OR1, OR2 and OR3 is equally critical to the switch because it mediates opposing regulation of divergent promoters. Site specificity at this second level is less well understood. Conformational adaptation by both the repressor and the different operators appears to be important. To evaluate how subunit-subunit interactions are involved in this process, we investigated the effects on both dimer stability and operator binding of amino acid substitutions at the contacts between the symmetrically related helices-5 in the dimer interface. Substitutions for Tyr88 alter dimer stability and greatly perturb differential operator affinity, but generally do not affect operator versus non-operator specificity. The pattern of these effects suggests that the geometry of the face-to-face aromatic stacking interaction between symmetrically related Tyr88 in each subunit, a group in the dimer interface but far removed from the DNA binding interface, plays a critical role in operator discrimination. Conformational changes in the tertiary structure of the subunits appears to be involved. By contrast, the significant effect of I84S substitution is to greatly decrease affinity for all three operators. Presumably, the altered packing of the dimer interface causes a quarternary structural change that moves the two helix-turn-helix motifs out of register with successive DNA major grooves.