Miyao T, Barnett J D, Woychik N A
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854-5635, USA.
J Biol Chem. 2001 Dec 7;276(49):46408-13. doi: 10.1074/jbc.M107012200. Epub 2001 Sep 27.
We used whole genome expression analysis to investigate the changes in the mRNA profile in cells lacking the Saccharomyces cerevisiae RNA polymerase II subunit RPB4 (Delta RPB4). Our results indicated that an essentially complete shutdown of transcription occurs upon temperature shift of this conditionally lethal mutant; 98% of mRNA transcript levels decrease at least 2-fold, 96% at least 4-fold. This data was supported by in vivo experiments that revealed a rapid and greater than 5-fold decline in steady state poly(A) RNA levels after the temperature shift. Expression of several individual genes, measured by Northern analysis, was also consistent with the whole genome expression profile. Finally we demonstrated that the loss of RNA polymerase II activity causes secondary effects on RNA polymerase I, but not RNA polymerase III, transcription. The transcription phenotype of the Delta RPB4 mutant closely mirrors that of the temperature-sensitive rpb1-1 mutant frequently implemented as a tool to inactivate the RNA polymerase II in vivo. Therefore, the Delta RPB4 mutant can be used to easily design strains that enable the study of distinct post-transcriptional cellular processes in the absence of RNA polymerase II transcription.
我们使用全基因组表达分析来研究缺乏酿酒酵母RNA聚合酶II亚基RPB4（ΔRPB4）的细胞中mRNA谱的变化。我们的结果表明，这种条件致死突变体在温度转变时会发生转录基本完全关闭；98%的mRNA转录水平至少下降2倍，96%至少下降4倍。体内实验支持了这一数据，该实验显示温度转变后稳态多聚腺苷酸（poly(A)）RNA水平迅速下降且下降幅度超过5倍。通过Northern分析测量的几个单个基因的表达也与全基因组表达谱一致。最后，我们证明RNA聚合酶II活性的丧失会对RNA聚合酶I的转录产生次级影响，但对RNA聚合酶III的转录没有影响。ΔRPB4突变体的转录表型与经常用作体内使RNA聚合酶II失活工具的温度敏感型rpb1-1突变体的转录表型非常相似。因此，ΔRPB4突变体可用于轻松设计菌株，以便在没有RNA聚合酶II转录的情况下研究不同的转录后细胞过程。
