Tetracycline-aptamer-mediated translational regulation in yeast
Tetracycline-aptamer-mediated translational regulation in yeast
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DOI：
10.1046/j.1365-2958.2003.03656.x
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发表时间：
2003-09-01
影响因子：
3.6
通讯作者：
Suess, B
中科院分区：
文献类型：
--
作者：
Hanson, S;Berthelot, K;Suess, B
我们描述了基于直接rna -配体相互作用的酵母转录后基因调控。四环素依赖的翻译调控可以通过在5‘非翻译区（5’ utr）的两个不同位置插入特定的适体来实施。加入四环素后，体内翻译被抑制了9倍。通过位于5'UTR起始密码子（cap-远端）附近的适体进行抑制比通过cap-近端位置进行抑制更有效。另一方面，在无细胞系统中抑制达到最大50倍，并且通过帽端适体最有效。对四环素依赖性翻译抑制的体外动力学研究表明，在开始翻译之前，四环素和mRNA的预孵育不仅导致了最快的抑制开始，而且是最有效的抑制。体内和体外调节系统行为的差异可能与完整细胞中mRNP结构和mRNA可及性的不同特性有关，而不是细胞提取物。在适体上游插入uORF序列后，也观察到四环素依赖性调节，表明我们的系统也靶向重新启动核糖体。多染色体梯度分析提供了对调控机制的深入了解。cap -近端插入抑制43S复合物与cap结构的结合，而start-密码子-近端适配体可能通过阻断扫描起始前复合物而干扰80S核糖体的形成。
We describe post-transcriptional gene regulation in yeast based on direct RNA-ligand interaction. Tetracycline-dependent translational regulation could be imposed via specific aptamers inserted at two different positions in the 5' untranslated region (5'UTR). Translation in vivo was suppressed up to ninefold upon addition of tetracycline. Repression via an aptamer located near the start codon (cap-distal) in the 5'UTR was more effective than repression via a cap-proximal position. On the other hand, suppression in a cell-free system reached maximally 50-fold and was most effective via a cap-proximal aptamer. Examination of the kinetics of tetracycline- dependent translational inhibition in vitro revealed that preincubation of tetracycline and mRNA before starting translation led not only to the fastest onset of inhibition but also the most effective repression. The differences between the behaviour of the regulatory system in vivo and in vitro are likely to be related to distinct properties of mRNP structure and mRNA accessibility in intact cells as opposed to cell-extracts. Tetracycline-dependent regulation was also observed after insertion of an uORF sequence upstream of the aptamer, indicating that our system also targets reinitiating ribosomes. Polysomal gradient analyses provided insight into the mechanism of regulation. Cap-proximal insertion inhibits binding of the 43S complex to the cap structure whereas start-codon-proximal aptamers interfere with formation of the 80S ribosome, probably by blocking the scanning preinitiation complex.