RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli
RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli
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DOI：
10.1111/j.1365-2958.2004.04258.x
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发表时间：
2004-10-01
影响因子：
3.6
通讯作者：
Drolet, M
中科院分区：
文献类型：
--
作者：
Baaklini, I;Hraiky, C;Drolet, M
人们早就知道，缺乏拓扑异构酶 I（topA 无效突变体）的大肠杆菌细胞无法生长。由于降低 DNA 旋转酶活性的突变以及由此产生的负超螺旋是为了补偿 topA 功能的丧失而发生的，因此人们假设过度的负超螺旋在某种程度上参与了 topA 无效突变体的生长抑制。然而，过度的负超螺旋如何抑制生长仍不清楚。我们之前已经证明，RNase HI（一种降解 R 环 RNA 部分的酶）的过量产生可以部分补偿由于拓扑异构酶 I 缺失而导致的生长缺陷。在本文中，我们研究了促旋酶重新激活对活跃生长的 topA 无效细胞的生理学影响。我们发现，旋转酶重新激活后，生长立即几乎完全停止，除非 RNase HI 过量产生。 Northern 印迹分析表明，当 RNase HI 没有过量产生时，细胞积累全长 mRNA 的能力显着降低。有趣的是，当缺乏 RNase HI 活性的细菌细胞以相同方式生长和处理时，也会出现类似的表型，尽管不太严重。总而言之，我们的结果表明，过度的负超螺旋会促进 R 环的形成，进而抑制 RNA 合成。
It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis.