Huet J, Sentenac A
Proc Natl Acad Sci U S A. 1987 Jun;84(11):3648-52. doi: 10.1073/pnas.84.11.3648.
The factor TUF interacts specifically with RPG or HOMOL1 sequences, which are present upstream of many genes coding for the yeast translational apparatus. Here we present evidence that the RPG and HOMOL1 motifs are variants of a consensus UASrpg (upstream activating sequence) recognized by the same factor. Factor TUF was identified by using two highly selective methods. The DNA-protein complex was isolated by pore-limit electrophoresis in polyacrylamide gradient gels and found to contain a single polypeptide of 150 kDa. In a two-step protein-blotting/nuclease-protection ("footprinting") procedure, the same 150-kDa polypeptide blotted on nitrocellulose exhibited the same specific DNA-binding properties as TUF factor. A 50-kDa DNA-binding domain of TUF was isolated by selective proteolysis. This suggests a bipolarization of the TUF protein, with distinct functional domains.
因子TUF与RPG或HOMOL1序列特异性相互作用,这些序列存在于许多编码酵母翻译装置的基因上游。在此我们提供证据表明,RPG和HOMOL1基序是被同一因子识别的共有UASrpg(上游激活序列)的变体。因子TUF是通过两种高度选择性的方法鉴定出来的。通过在聚丙烯酰胺梯度凝胶中进行孔径限制电泳分离出DNA-蛋白质复合物,发现其含有一条150 kDa的单一多肽。在两步蛋白质印迹/核酸酶保护(“足迹法”)程序中,印迹在硝酸纤维素膜上的同一条150 kDa多肽表现出与TUF因子相同的特异性DNA结合特性。通过选择性蛋白酶解分离出了TUF的一个50 kDa DNA结合结构域。这表明TUF蛋白具有双极化特性,带有不同的功能结构域。