Structural and mutational studies of the catalytic domain of colicin E5: A tRNA-specific ribonuclease
Structural and mutational studies of the catalytic domain of colicin E5: A tRNA-specific ribonuclease
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DOI：
10.1021/bi050749s
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发表时间：
2005-08-09
期刊：
影响因子：
2.9
通讯作者：
Huang, RH
中科院分区：
文献类型：
--
作者：
Lin, YL;Elias, Y;Huang, RH
Colicin E5 特异性切割大肠杆菌中的 4 个 tRNA，这些 tRNA 在摆动位置含有修饰的核苷酸 queuosine (Q)，从而阻止蛋白质合成并最终导致细胞死亡。在此，以 1.5 埃的分辨率测定了大肠杆菌中的大肠菌素 E5 (E5-CRD) 催化结构域的晶体结构。出乎意料的是，E5-CRD 采用了一种核心折叠，其中四链 β 折叠围绕 α 螺旋堆积，这在经过充分研究的核糖核酸酶 T1 中可见，尽管缺乏序列相似性。除了核心催化结构域外，N 端螺旋、C 端 β 链和环以及延伸的内环构成了 RNA 结合裂口。突变分析确定了 5 个对于 tRNA 底物结合和 E5-CRD 切割非常重要的氨基酸。该结构与突变研究一起使我们能够提出大肠菌素 ES-tRNA 相互作用的模型，表明 tRNA 底物识别的分子基础以及大肠菌素 E5 切割 tRNA 的机制。
Colicin E5 specifically cleaves four tRNAs in Escherichia coli that contain the modified nucleotide queuosine (Q) at the wobble position, thereby preventing protein synthesis and ultimately resulting in cell death. Here, the crystal structure of the catalytic domain of colicin E5 (E5-CRD) from E. coli was determined at 1.5 angstrom resolution. Unexpectedly, E5-CRD adopts a core folding with a four-stranded beta-sheet packed against an a-helix, seen in the well-studied ribonuclease T1 despite a lack of sequence similarity. Beyond the core catalytic domain, an N-terminal helix, a C-terminal beta-strand and loop, and an extended internal loop constitute an RNA binding cleft. Mutational analysis identified five amino acids that were important for tRNA substrate binding and cleavage by E5-CRD. The structure, together with the mutational study, allows us to propose a model of colicin ES-tRNA interactions,- suggesting the molecular basis of tRNA substrate recognition and the mechanism of tRNA cleavage by colicin E5.