Bredow S, Kleinert H, Benecke B J
Department of Biochemistry, Ruhr University, Bochum, F.R.G.
Gene. 1990 Feb 14;86(2):217-25. doi: 10.1016/0378-1119(90)90282-v.
We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.
我们已经在体外对S100提取物中RNA聚合酶III（RNAPIII）转录功能性人类7SL基因的情况进行了分析。7S L RNA的准确高效合成取决于以下两个条件：（i）一个上游序列；（ii）位于该基因前22个碱基对（bp）内的一个内部启动子元件。这些发现通过DNA酶I足迹法得到了证实。内部启动子的突变确定了A盒同源序列（核苷酸[nt]+5至+14）之外的CG双峰（nt+15/+16）对于体外转录试验中的正常启动子功能以及模板排除试验中的竞争均至关重要。对S100提取物进行分级分离后发现，除了RNAPIII之外，该基因的忠实转录还需要另外两个组分。在DNA酶I保护实验中观察到的两种足迹分别由这两个组分中的一个产生，这表明至少涉及两种DNA结合因子。
