T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates
T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates
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DOI：
10.1017/s135583829800106x
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发表时间：
1998-10-01
期刊：
影响因子：
4.5
通讯作者：
Uhlenbeck, OC
中科院分区：
文献类型：
--
作者：
Pleiss, JA;Derrick, ML;Uhlenbeck, OC
利用T7RNA聚合酶大量制备特定序列的RNA，极大地方便了对RNA结构和功能的研究。一般来说，人们认为这项技术的产物在序列上是高度同质的，只有少数值得注意的例外。我们仔细研究了几个5‘端序列不同的tRNA的转录产物，发现对于那些以多个连续的鸟苷开始的分子，转录产物远不是同源的。尽管以GCG开始的模板没有显示出可检测到的5‘端异质性，但设计为在其5’端具有四个或五个连续鸟苷的两个tRNA模板的总转录产物的30%以上在其5‘端延伸了至少一个非模板核苷酸。通过简单地减少连续鸟苷的数量，异质性显著降低。这种5‘端的异质性与T7转录中常见的3’端的异质性相结合，即使在严格的大小纯化后也会导致RNA分子的混合。
The use of T7 RNA polymerase to prepare large quantities of RNA of a particular sequence has greatly facilitated the study of both the structure and function of RNA. Generally, it has been believed that the products of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5' end sequence and found that, for those molecules that begin with multiple, consecutive guanosines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5' end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 5' ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 5' end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5' end heterogeneity in combination with the 3' end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification.