Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706.
Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI 53706.
Proc Natl Acad Sci U S A. 2019 Apr 23;116(17):8310-8319. doi: 10.1073/pnas.1819682116. Epub 2019 Apr 10.
The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) in , but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.
第二信使核苷酸 ppGpp 显著改变了细菌中的基因表达，以调节细胞代谢以适应营养物质的可用性。ppGpp 结合到 RNA 聚合酶 (RNAP) 上的两个位点上，但也有报道称它结合到许多其他蛋白质上。为了确定 RNAP 结合位点在 ppGpp 对转录的全基因组效应中的作用，我们使用 RNA-seq 分析了在缺乏/存在 RNAP 上的 ppGpp 结合位点的菌株中，响应升高的 ppGpp 水平产生的转录物。我们在没有伴随营养饥饿的情况下，在 ppGpp 产生后立即检查 RNA。此程序富集了 ppGpp 对 RNAP 的直接影响，而不是由于代谢饥饿引起的转录间接影响，或由于对 RNAP 的初始影响引起的二级事件。在 ppGpp 诱导 5 分钟后鉴定的所有 757 个基因的转录反应都依赖于 ppGpp 与 RNAP 的结合。大多数 (>75%)在早期研究中没有报道。调节的转录本编码的产物不仅参与翻译，还参与许多其他细胞过程。来自体内数据集的 100 多个启动子的体外转录分析确定了一大批直接调控的启动子，明确证明了 ppGpp 对体内转录的大多数影响是直接的，并允许比较受抑制、激活和不受影响的启动子类别的 DNA 序列。我们的分析大大扩展了我们对严格反应广度的理解，并提出了促进 ppGpp 特定影响的启动子序列特征。
