Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli.
Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli.
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转录RNA聚合酶和拓扑异构酶I之间的相互作用阻止了E.杆菌
DOI：
10.1038/s41467-022-32106-5
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发表时间：
2022-08-04
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
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作者：
细菌拓扑异构酶I(Topoi)可以去除过度的负超卷曲，并被认为在转录、复制和其他过程中放松DNA分子。利用CHIP-Seq，我们证明了大肠杆菌TOPOI(EcTopo I)与转录RNA聚合酶(RNAP)是共定位的。用转录延长抑制剂利福平处理会导致ECTOOI重新定位到启动子区域，在那里RNAP也会积累。当14 kDa RNAP结合的ECTOOI C末端结构域过表达时，ECTOOI和RNAP沿转录单位的共存减少。下拉实验直接证明这两种酶在体内相互作用。利用CHIP-SEQ和TOPO-SEQ，我们证明了ECTOOI富含在高活性转录单位的上游(高达12-15 kb)，表明ECTOOI松弛了转录产生的负超螺旋。RNAP：通过过表达ECTOOI竞争者(CTD或失活的ECTOOI Y319F突变体)或删除参与相互作用的ECTOOI结构域来解偶联：ECTOOI相互作用对细胞是有毒的，并导致过度的负质粒超螺旋。此外，RNAP：EcTopo I相互作用的解偶联导致全基因组R-环的积累，表明这种相互作用是防止R-环形成所必需的。在大肠杆菌中，Topoi和RNAP相互作用的中断降低了细胞的活力，导致DNA超负卷曲和R环积累。Topoi和DNA旋转酶结合在转录单位周围，Topoi通过特定的基序和负超螺旋识别切割位点。
Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is colocalized, genome-wide, with transcribing RNA polymerase (RNAP). Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, colocalization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched upstream (within up to 12-15 kb) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP:EcTopoI interaction by either overexpression of EcTopoI competitor (CTD or inactive EcTopoI Y319F mutant) or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, uncoupling of the RNAP:EcTopoI interaction leads to R-loops accumulation genome-wide, indicating that this interaction is required for prevention of R-loops formation. In E. coli, disruption of TopoI and RNAP interaction decreases cells viability and leads to hypernegative DNA supercoiling and R loops accumulation. TopoI and DNA gyrase bind around transcription units and TopoI recognizes cleavage sites by a specific motif and negative supercoiling.
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影响因子：
3.2
作者：
Baaklini, Imad;Usongo, Valentine;Drolet, Marc
通讯作者：
Drolet, Marc
影响因子：
14.9
作者：
Ashley RE;Dittmore A;McPherson SA;Turnbough CL Jr;Neuman KC;Osheroff N
通讯作者：
Osheroff N
影响因子：
4.5
作者：
El Sayyed, Hafez;Le Chat, Ludovic;Espeli, Olivier
通讯作者：
Espeli, Olivier
影响因子：
3.6
作者：
Baaklini, I;Hraiky, C;Drolet, M
通讯作者：
Drolet, M
影响因子：
--
作者：
Annamalai T;Dani N;Cheng B;Tse-Dinh YC
通讯作者：
Tse-Dinh YC