Shinya E, Shimada T
Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Nucleic Acids Res. 1994 Jun 11;22(11):2143-9. doi: 10.1093/nar/22.11.2143.
The human dihydrofolate reductase (DHFR) gene and mismatch repair protein 1 (MRP1) genes are organized in a head-to-head configuration separated by an 90 base pair sequence. We have previously shown that as small as a 114 bp promoter sequences is sufficient for accurate and efficient initiation of divergent transcription. In this study, the mechanism of accurate transcription initiation in vivo from this short bidirectional promoter was analyzed by a newly developed highly sensitive primer extension assay. The GC boxes in the middle of this sequence were essential for bidirectional promoter activity, but not sufficient for accurate initiation. The sequences overlapping the transcription initiation sites of the DHFR and MRP1 genes were shown to function as the initiator, which directs transcription from an internal site. These initiators were strictly position dependent and were active only when located from 40 to 50 base pairs downstream from the GC box. Although there is no apparent sequence homology between two initiators, a common nuclear factor bound to these elements. Existence of two initiators located on both sides of the middle GC box seems to be the molecular basis of bidirectional activity of this short DNA sequence.
人类二氢叶酸还原酶（DHFR）基因和错配修复蛋白1（MRP1）基因以头对头的方式排列，中间间隔90个碱基对序列。我们之前已经表明，小至114 bp的启动子序列就足以准确高效地启动双向转录。在本研究中，通过新开发的高灵敏度引物延伸试验分析了从这个短双向启动子在体内准确转录起始的机制。该序列中间的GC框对双向启动子活性至关重要，但不足以实现准确起始。与DHFR和MRP1基因转录起始位点重叠的序列显示可作为起始子，它指导从内部位点进行转录。这些起始子严格依赖位置，并且仅当位于GC框下游40至50个碱基对时才有活性。尽管两个起始子之间没有明显的序列同源性，但有一个共同的核因子与这些元件结合。位于中间GC框两侧的两个起始子的存在似乎是这个短DNA序列双向活性的分子基础。
