Linkage between substrate recognition and catalysis during cleavage of sarcin/ricin loop RNA by restrictocin
Linkage between substrate recognition and catalysis during cleavage of sarcin/ricin loop RNA by restrictocin
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DOI：
10.1021/bi700931y
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发表时间：
2007-11-06
期刊：
影响因子：
2.9
通讯作者：
Piccirilli, Joseph A.
中科院分区：
文献类型：
--
作者：
Korennykh, Alexei V.;Plantinga, Matthew J.;Piccirilli, Joseph A.
限制素是一种位点特异性核糖核酸内切酶，通过切割 23S-28S rRNA 的八叠球菌素/蓖麻毒素环 (SRL) 来灭活核糖体。在这里，我们基于监测 RNA 寡核苷酸（2-27-mers）的裂解，对 SRL 裂解反应进行动力学和热力学分析。限制素与 27 聚体 SRL 模型底物（通过静电相互作用指定为野生型 SRQ，形成非特异性基态复合物 E:S）结合。在 pH 6.7 时，物理步骤控制反应速率：野生型底物以部分扩散限制速率反应，而反应更快的 SRL（在可裂解磷酸盐处含有 3'-硫原子）以完全扩散限制速率反应(k(2)/K-1/2 = 1.1 x 10(9) M-1 s(-1))。在非特异性结合步骤之后，化学步骤明显限制了 SRL 切割率，该结构相对于单链 RNA 具有 4.3 kcal/mol 的过渡态稳定性。两个保守的 SRL 模块，凸出的 G 基序和 GAGA 四环，这些发现表明 SRL 识别模型，其中限制菌素接触 GAGA 四环和凸出的 G 基序的凸出鸟苷，从非特异性基态复合物 (E:S) 发展到更高能量特异性复合物 (E-S)，并在化学过渡态的过程中。对单链 RNA 的核糖核酸酶活性比限制素同系物、非结构特异性核糖核酸酶 T1 和 U2 小 10(3)-10(6) 倍，这些发现共同表明了 SRL 底物的结构特征如何促进催化，并提供了区分同源 RNA 和非同源 RNA 的机制。
Restrictocin is a site-specific endoribonuclease that inactivates ribosomes by cleaving the sarcin/ ricin loop (SRL) of 23S-28S rRNA. Here we present a kinetic and thermodynamic analysis of the SRL cleavage reaction based on monitoring the cleavage of RNA oligonucleotides (2-27-mers). Restrictocin binds to a 27-mer SRL model substrate (designated wild-type SRQ via electrostatic interactions to forin a nonspecific ground state complex E:S. At pH 6.7, physical steps govern the reaction rate: the wild-type substrate reacts at a partially diffusion-limited rate, and a faster-reacting SRL, containing a 3'-sulfur atom at the scissile phosphate, reacts at a fully diffusion-limited rate (k(2)/K-1/2 = 1.1 x 10(9) M-1 s(-1)). At pH 7.4, the chemical step apparently limits the SRL cleavage rate. After the nonspecific binding step, restrictocin recognizes the SRL structure, which imparts 4.3 kcal/mol transition state stabilization relative to a singlestranded RNA. The two conserved SRL modules, bulged-G motif and GAGA tetraloop, contribute at least 2.4 and 1.9 kcal/mol, respectively, to the recognition. These findings suggest a model of SRL recognition in which restrictocin contacts the GAGA tetraloop and the bulged guanosine of the bulged-G motif to progress from the nonspecific ground state complex (E:S) to the higher-energy-specific complex (E-S) en route to the chemical transition state. Comparison of restrictocin with other ribonucleases revealed that restrictocin exhibits a 10(3)-10(6)-fold smaller ribonuclease activity against single-stranded RNA than do the restrictocin homologues, non-structure-specific ribonucleases T1 and U2. Together, these findings show how structural features of the SRL substrate facilitate catalysis and provide a mechanism for distinguishing between cognate and noncognate RNA.