Model for RNA binding and the catalytic site of the RNase kid of the bacterial parD toxin-antitoxin system
Model for RNA binding and the catalytic site of the RNase kid of the bacterial parD toxin-antitoxin system
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DOI：
10.1016/j.jmb.2005.12.033
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发表时间：
2006-03-17
影响因子：
5.6
通讯作者：
Boelens, R
中科院分区：
文献类型：
--
作者：
Kamphuis, MB;Bonvin, AMJJ;Boelens, R
毒素Kid和抗毒素Kis由大肠杆菌质粒RI的parD操纵子编码。Kid及其染色体同源物MazF和ChpBK已显示抑制细胞提取物中的蛋白质合成，并在体外充当核糖体非依赖性核糖核酸内切酶。Kid优先在单链区域的核苷酸序列5 ′-UA（A/C）-3 ′中的A残基的5 ′侧切割RNA。在这里，我们表明，RNA裂解的基德产生两个片段的2 '：3'-环磷酸基团和一个自由的5 '-OH基团，分别。切割机制与RNA酶A和T1相似，涉及尿嘧啶2 '-OH基团。通过NMR滴定研究与所有不可切割的RNA模拟物，我们证明，Kid二聚体的两个单体的残基一起形成一个级联的RNA结合表面。基于NMR化学位移的对接计算、切割机制和先前报道的诱变数据提供了结合口袋内AUACA片段的位置的详细图片。我们提出残基D 75，R73和H17形成Kid毒素的活性位点，其中D 75和R73分别是催化碱和酸。RNA序列特异性由残基T46、S47、A55、F57、T69、V71和R73定义。我们的数据显示了这些残基对Kid功能的重要性，并讨论了我们的结果对相关毒素，如MazF，CcdB和RelE的影响。(c)2005爱思唯尔有限公司保留所有权利。
The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid RI. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-OH group. Via NMR titration studies With all uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, Such as MazF, CcdB and RelE, are discussed. (c) 2005 Elsevier Ltd. All rights reserved.