The Mechanisms of RNA SHAPE Chemistry
The Mechanisms of RNA SHAPE Chemistry
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DOI：
10.1021/ja2104075
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发表时间：
2012-04-18
影响因子：
15
通讯作者：
Weeks, Kevin M.
中科院分区：
文献类型：
--
作者：
McGinnis, Jennifer L.;Dunkle, Jack A.;Weeks, Kevin M.
RNA的生物学功能最终是由每个核苷酸的局部环境决定的。通过引物延伸（SHAPE）化学分析选择性2'-羟基酰化是测量不同生物环境中核苷酸结构和动力学的有力方法。SHAPE试剂对柔性核苷酸上的2'-羟基进行酰基化，因为不受约束的核苷酸优先取样增强2'-羟基亲核性的罕见构象。关键的推论是，一些受约束的核苷酸必须在2'-羟基上进行有效的反应。为了鉴定这些核苷酸，我们对大肠杆菌核糖体的完整晶体进行了SHAPE，监测了1490个核苷酸在16S rRNA中的反应性，并检查了那些对SHAPE具有高反应性且具有明确晶体结构的核苷酸。对这些构象的分析表明，涉及多个RNA官能团的一般碱催化和桥接3'-磷酸基团的两个特定取向广泛促进了2'-羟基的反应性。核苷酸类似物研究证实了这些机制对SHAPE反应性的贡献。这些结果为SHAPE反应性和局部RNA动力学之间的关系提供了强有力的机制解释，并将有助于在利用这种化学的许多技术中解释SHAPE信息。
The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2'-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2'-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2'-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the Escherichia coli ribosome, monitored the reactivity of 1490 nucleotides in 16S rRNA, and examined those nucleotides that were hyper-reactive toward SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2'-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3'-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry.