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A fluorescent assay for the genetic dissection of the RNA polymerase II termination machinery
Methods ( IF 4.2 ) Pub Date : 2019-04-01 , DOI: 10.1016/j.ymeth.2018.12.011
Daniel Reines 1
Affiliation  

RNA polymerase II is a highly processive enzyme that synthesizes mRNAs and some non-protein coding RNAs. Termination of transcription, which entails release of the transcript and disengagement of the polymerase, requires an active process. In yeast, there are at least two multi-protein complexes needed for termination of transcription, depending upon which class of RNAs are being acted upon. In general, the two classes are relatively short non-coding RNAs (e.g. snoRNAs) and relatively long mRNAs, although there are exceptions. Here, a procedure is described in which defective termination can be detected in living cells, resulting in a method that allows strains with mutations in termination factors or cis-acting sequences, to be identified and recovered. The strategy employs a reporter plasmid with a galactose inducible promoter driving transcription of green fluorescent protein which yields highly fluorescent cells. When a test terminator is inserted between the promoter and the fluorescent protein reading frame, cells fail to fluoresce. Mutant strains that have lost termination capability, so called terminator-override mutants, gain expression of the fluorescent protein and can be collected by fluorescence activated cell sorting. The strategy is robust since acquisition of fluorescence is a positive trait that has a low probability of happening adventitiously. Live mutant cells can easily be cloned from the population of positive candidates. Flow sorting is a sensitive, high-throughput detection step capable of discovering spontaneous mutations in yeast with high fidelity.

中文翻译:

用于 RNA 聚合酶 II 终止机制的遗传解剖的荧光测定

RNA 聚合酶 II 是一种高度加工酶,可合成 mRNA 和一些非蛋白质编码的 RNA。转录的终止需要一个活跃的过程,这需要转录物的释放和聚合酶的脱离。在酵母中,至少需要两种多蛋白复合物来终止转录,具体取决于所作用的 RNA 类别。通常,这两类是相对较短的非编码 RNA(例如 snoRNA)和相对较长的 mRNA,但也有例外。在这里,描述了一个程序,其中可以在活细胞中检测到有缺陷的终止,从而产生一种方法,可以识别和恢复具有终止因子或顺式作用序列突变的菌株。该策略采用具有半乳糖诱导型启动子的报告质粒,驱动绿色荧光蛋白的转录,产生高荧光细胞。当在启动子和荧光蛋白阅读框之间插入测试终止子时,细胞不会发出荧光。失去终止能力的突变株,即所谓的终止子覆盖突变体,获得荧光蛋白的表达,可以通过荧光激活细胞分选收集。该策略是稳健的,因为获得荧光是一种积极的特征,偶然发生的可能性很低。活的突变细胞可以很容易地从阳性候选细胞群中克隆出来。流式分选是一种灵敏、高通量的检测步骤,能够以高保真度发现酵母中的自发突变。
更新日期:2019-04-01
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