DiRusso C C, Metzger A K, Heimert T L
Department of Biochemistry, University of Tennessee, Memphis 38163.
Mol Microbiol. 1993 Jan;7(2):311-22. doi: 10.1111/j.1365-2958.1993.tb01122.x.
Fatty acid biosynthesis and fatty acid degradation in Escherichia coli are co-ordinately regulated at the level of transcription by the product of the fadR gene, FadR. In the present work we investigate FadR interaction with the fabA and fadL promoters. The FadR-responsive operator within fabA, OA, was localized to a region -47 to -31 base pairs relative to the start of transcription using DNase I protection studies. The promoter and untranslated leader within fadL had two binding sites for FadR, OL1 at -25 to -9 and OL2 at -1 to +16 relative to the start of transcription. The binding affinity of FadR for OA and OL1 or OL2 was lower than that for the single site within fadB (OB) as measured using protein-DNA gel retention assays. Overall, these experiments demonstrated that the affinity of FadR binding for DNA containing the fadB, fadL and fabA promoters was OB > OL1, OL2 > OA. We could not distinguish separate binding affinities for OL1 or OL2. We demonstrated repression of fadL transcription and activation of fabA transcription in vitro using run-off transcription assays containing purified FadR and RNA polymerase.
大肠杆菌中的脂肪酸生物合成和脂肪酸降解在转录水平上由fadR基因的产物FadR进行协调调控。在本研究中，我们研究了FadR与fabA和fadL启动子的相互作用。利用DNase I保护研究，fabA内的FadR反应性操纵子OA被定位到相对于转录起始点-47至-31碱基对的区域。fadL内的启动子和非翻译前导序列有两个FadR结合位点，相对于转录起始点，OL1在-25至-9，OL2在-1至+16。使用蛋白质-DNA凝胶滞留测定法测量，FadR对OA和OL1或OL2的结合亲和力低于对fadB内单个位点（OB）的结合亲和力。总体而言，这些实验表明，FadR与包含fadB、fadL和fabA启动子的DNA的结合亲和力为OB>OL1、OL2>OA。我们无法区分OL1或OL2的单独结合亲和力。我们使用含有纯化的FadR和RNA聚合酶的 runoff转录测定法在体外证明了fadL转录的抑制和fabA转录的激活。
