Uemura H, Koshio M, Inoue Y, Lopez M C, Baker H V
Department of Molecular Biology, National Institute of Bioscience and Human-Technology, Tsukuba Research Center, Ibaraki, Japan.
Genetics. 1997 Oct;147(2):521-32. doi: 10.1093/genetics/147.2.521.
To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and combinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-binding sites). The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene. As observed previously, synthetic oligonucleotides containing both CT- and RPG-boxes conferred strong UAS activity. Likewise, a lone CT-box did not show any UAS activity. By contrast, oligonucleotides containing tandem Ct-boxes but no RPG-box conferred strong promoter activity. This UAS activity was not dependent on position or orientation of the oligonucleotides in the 5' noncoding region. However, it was dependent on both GCR1 and GCR2. These results suggest that the ability of Gcr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent on Rap1p. Eleven independent mutants of GCR1 were isolated that conferred weak UAS activity to a single CT-box. Five mutants has single mutations in Gcr1p's DNA-binding domain and displayed slightly higher affinity for the CT-box. These results support the hypothesis that Gcr1p and Gcr2p play the central role in glycolytic gene expression and that the function of Rap1p is to facilitate the binding of Gcr1p to its target.
为了研究Gcr1p和Rap1p的相互依赖性，我们制备了一系列合成调控序列，这些序列包含不同数量和组合的CT-boxes（Gcr1p结合位点）和RPG-boxes（Rap1p结合位点）。使用ENO1-lacZ报告基因测试合成寡核苷酸作为调控序列的功能。如先前观察到的，同时包含CT-和RPG-boxes的合成寡核苷酸赋予了强大的上游激活序列（UAS）活性。同样，单独的CT-box没有显示出任何UAS活性。相比之下，含有串联Ct-boxes但没有RPG-box的寡核苷酸赋予了强大的启动子活性。这种UAS活性不依赖于寡核苷酸在5'非编码区的位置或方向。然而，它依赖于GCR1和GCR2。这些结果表明，Gcr1p在体内结合Gcr1p结合位点的能力并非绝对依赖于Rap1p。分离出11个独立的GCR1突变体，它们赋予单个CT-box弱UAS活性。5个突变体在Gcr1p的DNA结合结构域中有单个突变，并且对CT-box显示出略高的亲和力。这些结果支持以下假设：Gcr1p和Gcr2p在糖酵解基因表达中起核心作用，并且Rap1p的功能是促进Gcr1p与其靶标的结合。
