Shimada Tomohiro, Fujita Nobuyuki, Maeda Michihisa, Ishihama Akira
Nippon Institute for Biological Science, Division of Molecular Biology, Ome, Tokyo 198-0024, Japan.
Genes Cells. 2005 Sep;10(9):907-18. doi: 10.1111/j.1365-2443.2005.00888.x.
Cra (or FruR), a global transcription factor with both repression and activation activities, controls a large number of the genes for glycolysis and gluconeogenesis. To get insights into the entire network of transcription regulation of the E. coli genome by Cra, we isolated a set of Cra-binding sequences using an improved method of genomic SELEX. From the DNA sequences of 97 independently isolated DNA fragments by SELEX, the Cra-binding sequences were identified in a total of ten regions on the E. coli genome, including promoters of six known genes and four hitherto-unidentified genes. All six known promoters are repressed by Cra, but none of the activation-type promoters were cloned after two cyles of SELEX, because the Cra-binding affinity to the repression-type promoters is higher than the activation-type promoters, as determined by the quantitative gel shift assay. Of a total of four newly identified Cra-binding sequences, two are associated with promoter regions of the gapA (glyceraldehyde 3-phosphate dehydrogenase) and eno (enolase) genes, both involved in sugar metabolism. The regulation of newly identified genes by Cra was confirmed by the in vivo promoter strength assay using a newly developed TFP (two-fluorescent protein) vector for promoter assay or by in vitro transcription assay in the presence of Cra protein.
Cra（或FruR）是一种具有抑制和激活活性的全局转录因子，它控制着大量参与糖酵解和糖异生的基因。为了深入了解Cra对大肠杆菌基因组转录调控的整个网络，我们使用改进的基因组SELEX方法分离出了一组Cra结合序列。从通过SELEX独立分离的97个DNA片段的DNA序列中，在大肠杆菌基因组的总共十个区域中鉴定出了Cra结合序列，包括六个已知基因和四个迄今未鉴定基因的启动子。所有六个已知启动子均受Cra抑制，但在两轮SELEX后均未克隆到激活型启动子，因为通过定量凝胶迁移试验确定，Cra对抑制型启动子的结合亲和力高于激活型启动子。在总共四个新鉴定的Cra结合序列中，有两个与参与糖代谢的gapA（甘油醛-3-磷酸脱氢酶）和eno（烯醇化酶）基因的启动子区域相关。通过使用新开发的用于启动子分析的TFP（双荧光蛋白）载体进行体内启动子强度分析或在存在Cra蛋白的情况下进行体外转录分析，证实了Cra对新鉴定基因的调控。
