Estrem S T, Gaal T, Ross W, Gourse R L
Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706, USA.
Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9761-6. doi: 10.1073/pnas.95.17.9761.
The UP element, a component of bacterial promoters located upstream of the -35 hexamer, increases transcription by interacting with the RNA polymerase alpha-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 alpha 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 alpha-DNA interaction. Based on the evolutionary conservation of the residues in alpha 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.
UP元件是位于细菌启动子-35六聚体上游的一个组成部分，它通过与RNA聚合酶α亚基相互作用来增强转录。我们采用一种改进的SELEX程序来鉴定蛋白质结合位点，先在体外进行筛选，随后在体内筛选那些位于大肠杆菌rrnB P1核心启动子上游时能显著增强启动子活性的序列。其中一组31个这样的上游序列在体内使转录增加了136至326倍，比天然的rrnB P1 UP元件增加的幅度大得多，并用它们推导了共有序列：-59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn -38。最具活性的筛选序列包含推导的共有序列，具备UP元件的所有特性，且该序列与α亚基C末端结构域的相互作用类似于先前已鉴定的UP元件。UP元件共有序列的鉴定应有助于详细了解α亚基与DNA的相互作用。基于α亚基中负责与UP元件相互作用的残基的进化保守性，我们认为UP元件共有序列应适用于所有真细菌，通常有助于启动子预测，并且可能在生物技术应用中有用。
