Mechanisms of transcription fidelity in prokaryotes and eukaryotes

转录错误如何影响细胞生理学，以及细胞可以容忍何种程度的错误？对影响转录保真度的突变缺乏可靠的选择系统，阻碍了回答这些问题的进展。我们利用酿酒酵母RNA聚合酶II(POL II)进行了转录保真度控制的遗传和生化联合分析。我们选择并鉴定了POL II中一系列新的突变，使酵母对6-氮胞嘧啶敏感，这是一种使细胞内NTP池失衡的药物。Pol II催化Rpb1亚基E1103G突变是人工致死的，缺失了与RNA校对有关的DST1(转录裂解因子TFIIS)和RPB9(Pol II亚单位)基因，这意味着谷氨酸1103参与了转录保真度控制。在表达rpb1-E1103G等位基因的细胞中观察到的转录错误增加了几倍，证实了这一结论。我们在体外分析了突变的POL II，以确认观察到的突变的细胞表型是由容易出错的转录引起的，而不是由细胞生理或整体基因表达的变化引起的。Rpb1-E1103G使NTP掺入的保真度降低了至少7倍，在一些延长复合体中测试的保真度超过10倍，表明突变严重破坏了掺入前的保真度检查。即使在TFIIS存在的情况下，突变的聚合酶也表现出更高的错误传播机会，因为它增加了与下一个同源NTP的不匹配延伸的速率。我们提出，rpb1-E1103G和DST1缺失的合成致死性是由于基于Pol II和TFIIS的合作，同时消除了掺入前和掺入后的保真度控制而导致的错误突发。我们的结果提供了第一个证据，证明转录错误率在细胞内受到严格控制，保持转录保真度可能对细胞活力至关重要。

How do transcription errors affect cell physiology and what level of the errors can cells tolerate? Progress in answering these questions has been hampered by lack of a reliable selection system for mutations affecting transcription fidelity. We employed S. cerevisiae RNA polymerase II (Pol II) for a combined genetic and biochemical analyses of transcription fidelity control. We selected and characterized a series of novel mutations in Pol II rendering yeast sensitive to 6-azauracil, a drug that unbalances the intracellular NTP pool. Mutation E1103G of the catalytic Rpb1 subunit of Pol II was synthetic lethal with deletion of DST1 (transcript cleavage factor TFIIS) and RPB9 (subunit of Pol II) genes implicated in the RNA proofreading, which implicated glutamic acid 1103 in transcription fidelity control. This conclusion was reinforced by an observed several-fold increase in transcription errors in the cells expressing rpb1-E1103G allele. We analyzed the mutant Pol II in vitro to confirm that the observed cellular phenotypes of the mutations were caused by error-prone transcription rather than alterations in cell physiology or global gene expression. Rpb1-E1103G decreased fidelity of NTP incorporation at least seven-fold, and in a number of the elongation complexes tested over 10-fold indicating that the mutation severely damaged a pre-incorporation fidelity check. The mutant polymerase also exhibited a higher chance for error propagation even in the presence of TFIIS by increasing the rate of the mis-match extension with the next cognate NTP. We proposed that synthetic lethality of rpb1-E1103G and DST1 deletion was caused by an "error burst resulting from simultaneous elimination of pre- and post-incorporation fidelity controls based on the cooperation of Pol II and TFIIS. Our results provided the first evidence that transcription error rate is under a strict control in the cell and maintenance of transcription fidelity may be essential for cell viability.