Prevalence of RNA polymerase stalling at Escherichia coli promoters after open complex formation
Prevalence of RNA polymerase stalling at Escherichia coli promoters after open complex formation
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DOI：
10.1111/j.1365-2958.2008.06138.x
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发表时间：
2008-04-01
影响因子：
3.6
通讯作者：
Roberts, Jeffrey
中科院分区：
文献类型：
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作者：
Hatoum, Asma;Roberts, Jeffrey
RNA聚合酶（RNAP）被困在启动子逃逸的中间阶段，以及RNAP暂停在启动子近端sigma（70）依赖性暂停位点，产生稳定的，转录参与停滞的复合物，可以限制启动子功能和转录调控的潜在位点。为了调查这种中间体的患病率，我们筛选了118大肠杆菌候选启动子RNAP失速或附近的启动子，在体内KMnO4映射RNAP染色体DNA。在34个有活性的启动子中，前7个启动子lacZ、tnaA、cspA、cspD、rplK、rpsA和rpsU在体内具有停滞的RNAP，这一发现表明RNAP在启动后停滞在E.杆菌与流产和启动子近端sigma（70）依赖性暂停复合物的特征一致，在大多数新鉴定的失速位点处的RNAP捕获被rpoDL402F sigma（70）突变改变和位点突变消除，并且被GreA缺陷增强。除了启动子近端RNAP捕获，我们观察到转录依赖的DNA修饰跨越tnaA和cspA前导区的转录起始位点下游100 bp。
RNA polymerase (RNAP) trapped in intermediate stages of promoter escape, as well as RNAP paused at promoter-proximal sigma(70)-dependent pause sites, gives rise to stable, transcriptionally engaged stalled complexes that can limit promoter function and present potential sites for transcription regulation. To investigate the prevalence of such intermediates, we screened 118 Escherichia coli candidate promoters for RNAP stalling at or near the promoter, using in vivo KMnO4 mapping of RNAP on chromosomal DNA. Of 34 active promoters, the seven preceding lacZ, tnaA, cspA, cspD, rplK, rpsA and rpsU harboured stalled RNAP in vivo; this finding suggests that RNAP stalling after initiation is widespread in E. coli. Consistent with the characteristics of both abortive and promoter-proximal sigma(70)-dependent paused complexes, RNAP trapping at most of the newly identified stall sites was eliminated by the rpoDL402F sigma(70) mutational alteration and by site mutations, and was enhanced by GreA deficiency. In addition to promoter-proximal RNAP trapping, we observed transcription-dependent DNA modifications spanning the tnaA and cspA leader regions up to 100 bp downstream of the transcription start site.