Identification of an UP element consensus sequence for bacterial promoters

The UP element, a component of bacterial promoters located upstream of the −35 hexamer, increases transcription by interacting with the RNA polymerase α-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136- to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: −59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn −38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the α C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the α–DNA interaction. Based on the evolutionary conservation of the residues in α responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.

上游启动子元件（UP element）是细菌启动子中定位于−35六聚体上游的组件，可通过与RNA聚合酶α亚基相互作用提升转录效率。本研究采用经改良的指数富集配体系统进化技术（SELEX）流程以鉴定蛋白质结合位点，首先通过体外筛选获得候选序列，随后在体内验证：当此类序列置于大肠杆菌（Escherichia coli）rrnB P1核心启动子上游时，可显著提升启动子活性。其中31条上游序列可在体内将转录活性提升136至326倍，其增强效果远优于天然rrnB P1上游启动子元件；基于该批序列推导得到共有序列：−59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn −38。活性最强的筛选序列包含该推导所得的共有序列，且具备上游启动子元件的全部特征，其与α羧基末端结构域（α C-terminal domain）的相互作用模式与已报道的典型上游启动子元件一致。本次上游启动子元件共有序列的鉴定，将有助于深入解析RNA聚合酶α亚基与DNA的相互作用机制。鉴于负责结合上游启动子元件的α亚基残基在进化上具有保守性，我们认为该共有序列可推广应用于所有细菌域（eubacteria）生物，通常可助力启动子预测研究，且有望在生物技术领域得到应用。