Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme
Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme
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DOI：
10.1073/pnas.1414571111
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发表时间：
2014-09-09
影响因子：
11.1
通讯作者：
Steitz, Thomas A.
中科院分区：
文献类型：
--
作者：
Eiler, Daniel;Wang, Jimin;Steitz, Thomas A.
Twister是最近发现的一种RNA基序，据估计，它是已知催化速度最快的天然小分子自裂核酶之一。我们从一个非分离培养的生物体中确定了一个4.1埃分辨率的扭扭序列晶体结构，它显示了一个有序的可剪切磷酸和核苷酸5'到裂解位点。在3.1埃分辨率下测定的Orzyza sativa twister的第二种晶体结构显示出裂解位点的无序可裂磷酸盐和核苷酸5'。捻线的核心是通过碱基配对、一个大的堆叠相互作用网络和两个假结来稳定的。我们观察到三种核苷酸似乎介导催化作用：一种鸟苷，我们提出将核苷酸5‘的2’-羟基去质子化到裂解位点，另一种是保守的腺苷。我们认为腺苷在裂解位点中和了非桥接磷酸氧原子上的负电荷。活性位点还定位了不稳定的连锁，用于在线亲核攻击，因此twister似乎同时使用了三种针对小型自裂核酶提出的策略。扭扭体晶体结构(i)显示了其整体结构，（ii）证明了双假结褶皱的重要性，（iii）为增强催化提供了可能的假设，（iv）阐明了扭扭体所有10个高度保守的核苷酸参与其小而稳定的催化口袋形成的作用。
Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-angstrom resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5' to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-angstrom resolution exhibits a disordered scissile phosphate and nucleotide 5' to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2'-hydroxyl of the nucleotide 5' to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.