Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
Mol Cell. 2013 May 9;50(3):420-9. doi: 10.1016/j.molcel.2013.03.021. Epub 2013 Apr 25.
The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.
全球调节核苷酸 ppGpp（“魔点”）调节了大肠杆菌中很大一部分启动子的转录，这说明了小分子如何通过与 RNA 聚合酶 (RNAP) 相互作用而无需与 DNA 结合，特异性地控制基因表达。然而，ppGpp 在 RNAP 上的靶位点及其作用机制仍然不清楚。我们在这里报道了大肠杆菌 RNAP 上的 ppGpp 结合位点，该位点是通过交联、蛋白酶作图和分析不能对 ppGpp 做出响应的突变型 RNAP 鉴定出来的。一个带有突变 ppGpp 结合位点的菌株表现出与 ppGpp 合成缺陷细胞的特征。该结合位点位于两个 RNAP 亚基 ω 和 β'的界面上，其位置表明一种变构作用机制，涉及两个可移动的 RNAP 模块之间的运动限制。结合位点的鉴定允许通过靶向 RNAP 来预测 ppGpp 发挥作用的细菌种类。
