Mutational analysis of the Myxococcus xanthus Omicron4499 promoter region reveals shared and unique properties in comparison with other C-signal-dependent promoters

The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior. C-signal affects gene expression late in development, including that of Omega4499, an operon identified by insertion of Tn5 lac into the M. xanthus chromosome. The Omega4499 promoter region has several sequences in common with those found previously to be important for expression of other C-signal-dependent promoters. To determine if these sequences are important for Omega4499 promoter activity, the effects of mutations on expression of a downstream reporter gene were tested in M. xanthus. Although the promoter resembles those recognized by Escherichia coli sigma(54), mutational analysis implied that a sigma(70)-type sigma factor likely recognizes the promoter. A 7-bp sequence known as a C box and a 5-bp element located 6 bp upstream of the C box have been shown to be important for expression of other C-signal-dependent promoters. The Omega4499 promoter region has C boxes centered at -33 and -55 bp, with 5-bp elements located 7 and 8 bp upstream, respectively. A multiple-base-pair mutation in any of these sequences reduced Omega4499 promoter activity more than twofold. Single base-pair mutations in the C box centered at -33 bp yielded a different pattern of effects on expression than similar mutations in other C boxes, indicating that each functions somewhat differently. An element from about -81 to -77 bp exerted a twofold positive effect on expression but did not appear to be responsible for the C-signal dependence of the Omega4499 promoter. Mutations in sigD and sigE, which are genes that encode sigma factors, reduced expression from the Omega4499 promoter. The results provide further insight into the regulation of C-signal-dependent genes, demonstrating both shared and unique properties among the promoter regions so far examined.     

人A4GNT基因5'调控区的结构和功能分析

为探索人α1,4-N-乙酰葡糖胺转移酶(α1,4-N-acetylglucosaminyltransferase,A4GNT)基因表达的调控机制,应用5'cDNA末端快速扩增法和引物延伸法确定了A4GNT基因的转录起始位点.在生物信息学分析的基础上,构建了系列5'缺失荧光素酶报告基因载体和定点突变载体.瞬时转染胃癌细胞MKN45和AGS.荧光素酶活性分析表明,A4GNT基因转录的核心启动子在-141bp～+116bp区域,该区域缺乏典型的TATA盒,但含有CCAAT盒、Sp1和ETS-1等转录因子潜在结合位点.突变分析显示,-136bp～-131bp的Sp1结合位点及-93bp～-89bp正向CCAAT序列对A4GNT启动子转录激活至关重要.电泳迁移率变动分析表明,这两个顺式作用元件能够与转录因子Sp1和NF-Y结合.另外,在-1464bp～-771bp区域可能含有与基因的特异性表达相关的调控元件.