Fluorescent labeling of tRNAs for dynamics experiments
Fluorescent labeling of tRNAs for dynamics experiments
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DOI：
10.1261/rna.475407
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发表时间：
2007-09-01
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：
Betteridge, Thu;Liu, Hanqing;Hou, Ya-Ming
转运RNA（transfer RNA，tRNA）是氨酰-tRNA合成酶和核糖体等复杂酶的底物，在遗传信息转化为蛋白质序列的过程中起着重要作用。在这里，我们描述了一个通用的方法标记的tRNA与荧光染料，使标记的tRNA的活动和动力学可以直接监测荧光在核糖体解码过程中。该方法利用了先前报道的天然tRNA在二氢尿苷（D）位置的荧光标记，但通过制备tRNA转录物并使用酵母酶Dus 1 p二氢尿苷合酶将D残基引入转录物，将先前的方法扩展到合成tRNA。使用未经修改的转录大肠杆菌tRNA（Pro）作为一个例子，其中有U17和U17 a的D环，我们表明，Dus 1 p催化这些美国之一（主要是U17 a）转化为D，并修改后的tRNA可以标记的荧光团proflavin和罗丹明110，与整体标记产率与天然酵母tRNA（苯丙氨酸）。此外，转录酵母tRNA（苯丙氨酸），修改Dus 1 p和标记与proflavin，易位的核糖体上的速率类似的proflavin标记的天然酵母tRNA（苯丙氨酸）。这些结果表明，合成的tRNA转录本，它可以被设计为包含在自然界中没有发现的突变，可以被标记和研究。这种标记的tRNA应该在研究中具有广泛的用途，包括tRNA成熟，氨酰化和tRNA-核糖体相互作用的研究。
Transfer RNAs (tRNAs) are substrates for complex enzymes, such as aminoacyl-tRNA synthetases and ribosomes, and play an essential role in translation of genetic information into protein sequences. Here we describe a general method for labeling tRNAs with fluorescent dyes, so that the activities and dynamics of the labeled tRNAs can be directly monitored by fluorescence during the ribosomal decoding process. This method makes use of the previously reported fluorescent labeling of natural tRNAs at dihydrouridine ( D) positions, but extends the previous method to synthetic tRNAs by preparing tRNA transcripts and introducing D residues into transcripts with the yeast enzyme Dus1p dihydrouridine synthase. Using the unmodified transcript of Escherichia coli tRNA(Pro) as an example, which has U17 and U17a in the D loop, we show that Dus1p catalyzes conversion of one of these Us ( mostly U17a) to D, and that the modified tRNA can be labeled with the fluorophores proflavin and rhodamine 110, with overall labeling yields comparable to those obtained with the native yeast tRNA(Phe). Further, the transcript of yeast tRNA(Phe), modified by Dus1p and labeled with proflavin, translocates on the ribosome at a rate similar to that of the proflavin-labeled native yeast tRNA(Phe). These results demonstrate that synthetic tRNA transcripts, which may be designed to contain mutations not found in nature, can be labeled and studied. Such labeled tRNAs should have broad utility in research that involves studies of tRNA maturation, aminoacylation, and tRNA-ribosome interactions.