Modulation of tRNAAla identity by inorganic pyrophosphatase
Modulation of tRNAAla identity by inorganic pyrophosphatase
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DOI：
10.1073/pnas.092152799
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发表时间：
2002-04-30
影响因子：
11.1
通讯作者：
Uhlenbeck, OC
中科院分区：
文献类型：
--
作者：
Wolfson, AD;Uhlenbeck, OC
开发了一种高度灵敏的 tRNA 氨酰化检测方法，通过跟踪 3'-P-32 标记 tRNA 上的氨基酸附着情况，直接测量氨酰化 tRNA 的分数。当应用于大肠杆菌丙氨酰-tRNA 合成酶时，该测定可以准确测量最有害的 tRNA(Ala) 突变体的氨酰化。在无机焦磷酸酶和延伸因子 Tu 存在和不存在的情况下，评估了 tRNAAla 同一性突变对氨酰化效率 (k(cat)/K-M) 和氨酰-tRNA 稳态水平的影响。即使 k(cat)/K-M 值降低数千倍，tRNA 突变体仍能实现显着水平的氨酰化。这些结果部分地调和了 tRNA(Ala) 身份的体内和体外分析之间的差异。
A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3'-P-32-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA(Ala). The effect of tRNAAla identity mutations on both aminoacylation efficiency (k(cat)/K-M) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the k(cat)/K-M value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA(Ala) identity.