Bredow S, Sürig D, Müller J, Kleinert H, Benecke B J
Department of Biochemistry, Faculty of Chemistry, Ruhr-University, Bochum, FRG.
Nucleic Acids Res. 1990 Dec 11;18(23):6779-84. doi: 10.1093/nar/18.23.6779.
The gene-external part of the human 7S L promoter was analyzed by transcription in vitro and in vivo. Compared to the wild type promoter (-178), a -66 5'deletion mutant revealed full activity in vitro but was inefficiently transcribed in vivo. Further deletion to -37 reduced template activity to 50% in vitro and to basal level expression in vivo (below 5%). A DNase I footprint observed around position -50 protected an ATF-like binding site ('TGACGT'). With respect to 7S L transcription regulation, the functionality of this ATF-like binding site was confirmed in competition experiments and by mutation analysis. Furthermore, S100 extracts of cells pretreated with forskolin in vivo to induce the cAMP system, revealed significantly increased transcription of 7S L RNA in vitro, with no effect on a 7S K RNA gene, lacking such an ATF binding site. Thus, the 7S L RNA gene too is controlled by a regulatory element originally defined in class II promoters and represents another rare example where a specific type of transcription regulation in vivo can be mimicked with cell-free extracts in vitro.
通过体外和体内转录对人7S L启动子的基因外部区域进行了分析。与野生型启动子（-178）相比，-66 5'缺失突变体在体外显示出完全活性，但在体内转录效率低下。进一步缺失至-37会使模板活性在体外降至50%，在体内降至基础水平表达（低于5%）。在-50位置附近观察到的DNase I足迹保护了一个类似ATF的结合位点（“TGACGT”）。关于7S L转录调控，在竞争实验和突变分析中证实了这个类似ATF结合位点的功能。此外，体内用福斯高林预处理以诱导cAMP系统的细胞的S100提取物，在体外显示出7S L RNA转录显著增加，而对缺乏这种ATF结合位点的7S K RNA基因没有影响。因此，7S L RNA基因也受最初在II类启动子中定义的调控元件控制，并且代表了另一个罕见的例子，即体内特定类型的转录调控可以在体外无细胞提取物中模拟。
