Hypernegative Supercoiling Inhibits Growth by Causing RNA Degradation
Hypernegative Supercoiling Inhibits Growth by Causing RNA Degradation
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DOI：
10.1128/jb.00680-08
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发表时间：
2008-11-01
影响因子：
3.2
通讯作者：
Drolet, Marc
中科院分区：
文献类型：
--
作者：
Baaklini, Imad;Usongo, Valentine;Drolet, Marc
转录诱导的超负性超螺旋是大肠杆菌拓扑异构酶I（topA）突变体的标志。然而，其生理意义仍不清楚。温度下移的突变体产生短暂的生长停滞和平行增加的超负超螺旋，更严重的温度较低。这两种性质都通过过度表达RNase HI而减轻。虽然提取物中的核糖体表现出正常的活动时，在生长停滞，核糖体上的mRNA减少的FIS和CRP的较短，多聚核糖体相对于单体丰富得多，和蛋白质合成率下降，大，小蛋白质的比例一样。还观察到lacA和fis mRNA的加工和降解改变。这些数据与生长停滞期间mRNA的截短一致。这些效果不受编码RNase E的基因突变的影响，表明这种核酸内切酶不参与异常mRNA加工。它们也不受大观霉素的影响，大观霉素是一种蛋白质合成的抑制剂，它反对RNA酶活性的诱导。在体外转录显示，R-环的形成是更广泛的超负超螺旋模板。这些结果使我们首次提出了一个模型，通过该模型，超负超螺旋抑制生长。在该模型中，通过促旋酶引入超负超螺旋促进新生RNA的降解; RNA酶HI的过量产生限制了超负超螺旋的积累，从而防止了广泛的RNA降解。
Transcription-induced hypernegative supercoiling is a hallmark of Escherichia coli topoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA on ribosomes was reduced for fis and shorter for crp, polysomes were much less abundant relative to monosomes, and protein synthesis rate dropped, as did the ratio of large to small proteins. Altered processing and degradation of lacA and fis mRNA was also observed. These data are consistent with truncation of mRNA during growth arrest. These effects were not affected by a mutation in the gene encoding RNase E, indicating that this endonuclease is not involved in the abnormal mRNA processing. They were also unaffected by spectinomycin, an inhibitor of protein synthesis, which argued against induction of RNase activity. In vitro transcription revealed that R-loop formation is more extensive on hypernegatively supercoiled templates. These results allow us, for the first time, to present a model by which hypernegative supercoiling inhibits growth. In this model, the introduction of hypernegative supercoiling by gyrase facilitates degradation of nascent RNA; overproduction of RNase HI limits the accumulation of hypernegative supercoiling, thereby preventing extensive RNA degradation.