tRNA 3′-amino-tailing for stable amino acid attachment
tRNA 3′-amino-tailing for stable amino acid attachment
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DOI：
10.1261/rna.068015.118
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发表时间：
2018-12-01
期刊：
影响因子：
4.5
通讯作者：
Hou, Ya-Ming
中科院分区：
文献类型：
--
作者：
Gamper, Howard;Hou, Ya-Ming
氨基酸附着在tRNA 3'端，是进入核糖体进行蛋白质合成的先决条件。氨基酸附着也使tRNA能够进入非核糖体细胞活动。然而，正常的连接是通过氨基酸的羧基和tRNA末端A76核糖的3'-羟基之间的酯连接。这种酯链的不稳定性严重阻碍了氨基酰基trna的研究。尽管在3‘氨基末端tRNA中使用3’-氨基-3'-脱氧A76通过酰胺键提供稳定的氨基酰基连接，但存在多种尾随方案，每种方案相对于其他方案的效率尚不清楚。在这里，我们平行比较了五种不同的尾矿方案，所有这些方案都依赖于cca添加酶[CTP(ATP): tRNA核苷酸转移酶；（缩写为CCA酶）用修饰过的核糖交换天然核糖。我们发现，最有效的方案是通过cca催化的热磷酸化水解，在平衡状态下去除天然A76，并添加适当的ATP类似物来合成修饰的3'端。这个3'-氨基尾链的方案提供了广泛的氨基酸与tRNA的定量和稳定的附着，表明它在典型和非典型活性的氨基酰tRNA研究中具有普遍的实用性。
Amino acids are attached to the tRNA 3'-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3'-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3'-amino-3'-deoxy A76 in a 3'amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown. Here we compare five different tailing protocols in parallel, all dependent on the CCA-adding enzyme [CTP(ATP): tRNA nucleotidyl transferase; abbreviated as the CCA enzyme] to exchange the natural ribose with the modified one. We show that the most efficient protocol is achieved by the CCA-catalyzed pyrophosphorolysis removal of the natural A76 in equilibrium with the addition of the appropriate ATP analog to synthesize the modified 3'-end. This protocol for 3'-amino-tailing affords quantitative and stable attachment of a broad range of amino acids to tRNA, indicating its general utility for studies of aminoacyl-tRNAs in both canonical and noncanonical activities.