Aiyar S E, Gourse R L, Ross W
Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706, USA.
Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14652-7. doi: 10.1073/pnas.95.25.14652.
Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the alpha subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP alpha subunit. The A-tract sequence was protected by wild-type RNAP but not by alpha-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the -35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP alphaCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.
上游A序列可刺激多种细菌启动子的转录，这一点已被广泛归因于含A序列的DNA内在曲率的直接影响。在本研究中，我们报告了一些实验，这些实验提示了上游A序列影响转录的一种不同机制。含A序列与某些细菌启动子中富含腺嘌呤和胸腺嘧啶的上游识别元件（UP元件）的相似性表明，A序列可能通过与RNA聚合酶（RNAP）的α亚基相互作用来增加启动子活性。我们发现，置于大肠杆菌lac或rrnB P1启动子上游的含A序列在体内和体外均能刺激转录，且这种刺激需要RNAP α亚基的C末端（DNA结合）结构域。在足迹实验中，野生型RNAP可保护A序列，而α突变型RNAP则不能。A序列对转录的影响不如最活跃的UP元件大，这与A序列与UP元件共有序列的相似程度一致。当A序列靠近-35六聚体而非更上游一个螺旋圈的位置时，其功能最佳，这与UP元件功能的最佳定位相似。我们得出结论，A序列起到UP元件的作用，通过为RNAP αCTD提供结合位点来刺激转录，并且我们认为这些相互作用可能有助于先前描述的启动子DNA围绕RNAP的包裹。
