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：

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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。