Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop
Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop
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DOI：
10.1261/rna.1257509
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发表时间：
2009-02-01
期刊：
影响因子：
4.5
通讯作者：
Cooperman, Barry S.
中科院分区：
文献类型：
--
作者：
Pan, Dongli;Qin, Haiou;Cooperman, Barry S.
我们描述了一个优化的程序，用于取代二氢尿苷残基的收费tRNA与Cy 3和Cy 5染料连接到一个酰肼基团，并证明标记的分子是功能性的核糖体活动，包括30 S起始复合物的形成，EF-Tu依赖性结合到核糖体，易位，和多肽的合成。这个过程应该可以直接推广到其他酰肼连接的荧光团掺入tRNA或其他含二氢尿苷的RNA。此外，我们使用快速周转FRET实验，测量Cy 5-标记的tRNA（fMet）和Cy 3-标记的fMetPhe-tRNA（Phe）之间的能量转移，以获得直接的证据，支持易位的早期步骤涉及tRNA的柔性3 '-单链区域的运动的假设，与相当大的增加的距离分开的两个tRNA三级核心发生在该过程的后期。
We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3'-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.