Topological constraints impair RNA polymerase II transcription and causes instability of plasmid-borne convergent genes
Topological constraints impair RNA polymerase II transcription and causes instability of plasmid-borne convergent genes
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DOI：
10.1093/nar/gkr840
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发表时间：
2012-02-01
影响因子：
14.9
通讯作者：
Aguilera, Andres
中科院分区：
文献类型：
--
作者：
Garcia-Rubio, Maria L.;Aguilera, Andres
尽管拓扑异构酶和超卷曲变化与转录和复制之间存在关联的理论基础，但我们对拓扑约束对转录和复制的影响的了解尚不完整。虽然拓扑异构酶的突变会影响rDNA区域的表达和稳定性，但目前尚不清楚RNAPII的转录和其他区域的基因组完整性是否也会受到影响。我们开发了两个聚合rnapii驱动基因同时转录的新检测方法。基于质粒的系统在两个收敛的转录单元之间有转录终止子和没有转录终止子，这样也可以评估转录干扰的影响。通过这些实验，我们发现Topos I和Topos II在体内RNAPII转录中发挥作用，并降低了RNAPII转录基因在酿酒酵母中的稳定性。聚合转录单位中的超卷曲积累会损害top1 Delta菌株中RNAPII的转录，但Topo II也是独立于Topo I和可检测到的超卷曲积累的高效转录所必需的。我们的研究表明，拓扑限制会对RNAPII的转录和遗传完整性产生负面影响，并提供了一种通过转录干扰研究基因调控的方法。
Despite the theoretical bases for the association of topoisomerases and supercoiling changes with transcription and replication, our knowledge of the impact of topological constraints on transcription and replication is incomplete. Although mutation of topoisomerases affects expression and stability of the rDNA region it is not clear whether the same is the case for RNAPII transcription and genome integrity in other regions. We developed new assays in which two convergent RNAPII-driven genes are transcribed simultaneously. Plasmid-based systems were constructed with and without a transcription terminator between the two convergent transcription units, so that the impact of transcription interference could also be evaluated. Using these assays we show that Topos I and II play roles in RNAPII transcription in vivo and reduce the stability of RNAPII-transcribed genes in Saccharomyces cerevisiae. Supercoiling accumulation in convergent transcription units impairs RNAPII transcription in top1 Delta strains, but Topo II is also required for efficient transcription independent of Topo I and of detectable supercoiling accumulation. Our work shows that topological constraints negatively affect RNAPII transcription and genetic integrity, and provides an assay to study gene regulation by transcription interference.