Cohen R B, Yang L, Thompson J A, Safer B
Section on RNA and Protein Biosynthesis, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
J Biol Chem. 1988 Jul 25;263(21):10377-85.
Gel electrophoresis mobility shift and DNase I footprint assays detect a cellular nuclear protein in extracts made from uninfected human cells which binds to a downstream promoter sequence (DPS) in the human adenovirus 2 major late promoter. By DNase I footprint and mutation analyses, we have determined that this new regulatory element extends from positions +146 to +165 (relative to the cap site at position +1). We show by UV cross-linking that a 40-kDa polypeptide specifically binds to this region. Mutations within the DPS which decrease protein binding by 80-90% also cause a 2.5-3-fold decrease in in vitro major late promoter transcription efficiency. Alteration of the template in the 5'-flanking region of the DPS does not affect nuclear protein binding or transcription efficiency. Interestingly, a T----G transversion at position +160 which increases protein binding also impairs promoter activity.
凝胶电泳迁移率变动分析和DNase I足迹分析检测到未感染的人类细胞提取物中的一种细胞核蛋白，该蛋白可与人腺病毒2型主要晚期启动子中的下游启动子序列（DPS）结合。通过DNase I足迹分析和突变分析，我们确定这个新的调控元件从+146位延伸至+165位（相对于+1位的帽位点）。我们通过紫外线交联表明，一种40 kDa的多肽特异性结合该区域。DPS内的突变使蛋白质结合减少80 - 90%，同时也导致体外主要晚期启动子转录效率降低2.5 - 3倍。DPS 5'侧翼区域模板的改变不影响核蛋白结合或转录效率。有趣的是，+160位的T→G颠换增加了蛋白质结合，但也损害了启动子活性。
