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Author:
Date:
2014-09-20
[Abstract] This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the binding assay to mimic the “natural” situation in the bacterial cell, where the pre-formed, stable tRNA is already present while the T-box leader RNA is actively transcribed by the RNA polymerase. The ...
[摘要] 该方案描述了（i）"活"体外RNA转录与（ii）通过放射性标记的预先形成的tRNA的结合，然后是天然凝胶电泳和磷光成像仪扫描以显现复合物的偶联。 必要性来自一种RNA在不存在其相互作用配偶体时形成的稳定结构。 T盒前导RNA，转录控制系统，折叠成热力学非常稳定的茎 - 环结构，没有tRNA存在，这使得体外结合两个预先形成的RNA的相互作用非常困难。 因此，我调整结合测定以模拟细菌细胞中的"天然"情况，其中预先形成的稳定的tRNA已经存在，而T盒前导RNA被RNA聚合酶主动转录。 方案的第一部分还描述了体外转录和tRNA的标记。
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