Novel structural and functional mode of a knot essential for RNA binding activity of the Esa1 presumed chromodomain.
Novel structural and functional mode of a knot essential for RNA binding activity of the Esa1 presumed chromodomain.
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DOI：
10.1016/j.jmb.2008.03.021
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发表时间：
2008-05
影响因子：
5.6
通讯作者：
H. Shimojo;N. Sano;Yoshihito Moriwaki;M. Okuda;M. Horikoshi;Y. Nishimura
中科院分区：
文献类型：
--
作者：
H. Shimojo;N. Sano;Yoshihito Moriwaki;M. Okuda;M. Horikoshi;Y. Nishimura
染色体结构域是甲基化的组蛋白结合模块，已被广泛研究。有趣的是，据报道一些染色域可以与RNA和/或DNA结合，尽管它们的RNA/DNA相互作用的分子基础尚未得到解决。在这里，我们提出了一种新的染色体结构域-RNA相互作用的结合模式。Essential Sas-related acetyltransferase 1（Esa1）除了含有组蛋白乙酰转移酶结构域外，还含有一个假定的染色体结构域。我们最初确定了Esa 1假定的染色体结构域的溶液结构，并表明它由一个折叠良好的结构组成，含有一个类似于tudor结构域的五链β-桶，而不是典型的染色体结构域。此外，该结构域没有显示出RNA/DNA结合能力。由于蛋白质的N-末端形成螺旋转角，我们制备了N-末端延伸的构建体，我们惊讶地发现其与聚（U）结合并且对体内功能至关重要。这种延伸的蛋白质含有额外的β-折叠，其充当tudor结构域的结，并且与迄今为止检测的其他寡聚RNA和DNA相比以更大的亲和力结合寡聚（U）和寡聚（C）。结不会引起核心结构的整体变化，但会在tudor结构域本身诱导一个明确的环，这是RNA结合的原因。我们在酵母中的Esa 1突变基因中制造了47个点突变体，其中Esa 1打结的tudor结构域的氨基酸被替换为丙氨酸残基，并检测了它们的功能能力。有趣的是，对酵母致命的打结的tudor结构域突变失去了poly（U）结合能力。与RNA相互作用位点相关的氨基酸，如通过NMR和亲和结合实验所揭示的，被发现在体内是重要的。这些发现首次证明了打结的都铎结构域的新结构如何影响RNA结合以及这如何影响体内功能。
Chromodomains are methylated histone binding modules that have been widely studied. Interestingly, some chromodomains are reported to bind to RNA and/or DNA, although the molecular basis of their RNA/DNA interactions has not been solved. Here we propose a novel binding mode for chromodomain–RNA interactions. Essential Sas-related acetyltransferase 1 (Esa1) contains a presumed chromodomain in addition to a histone acetyltransferase domain. We initially determined the solution structure of the Esa1 presumed chromodomain and showed it to consist of a well-folded structure containing a five-stranded β-barrel similar to the tudor domain rather than the canonical chromodomain. Furthermore, the domain showed no RNA/DNA binding ability. Because the N-terminus of the protein forms a helical turn, we prepared an N-terminally extended construct, which we surprisingly found to bind to poly(U) and to be critical for in vivo function. This extended protein contains an additional β-sheet that acts as a knot for the tudor domain and binds to oligo(U) and oligo(C) with greater affinity compared with other oligo-RNAs and DNAs examined thus far. The knot does not cause a global change in the core structure but induces a well-defined loop in the tudor domain itself, which is responsible for RNA binding. We made 47 point mutants in an esa1 mutant gene in yeast in which amino acids of the Esa1 knotted tudor domain were substituted to alanine residues and their functional abilities were examined. Interestingly, the knotted tudor domain mutations that were lethal to the yeast lost poly(U) binding ability. Amino acids that are related to RNA interaction sites, as revealed by both NMR and affinity binding experiments, are found to be important in vivo. These findings are the first demonstration of how the novel structure of the knotted tudor domain impacts on RNA binding and how this influences in vivo function.