Toth M, Doerfler W, Shenk T
Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, NJ 08544-1014.
Nucleic Acids Res. 1992 Oct 11;20(19):5143-8. doi: 10.1093/nar/20.19.5143.
The activity of the adenovirus major late promoter is substantially increased as the infection proceeds from the early to late phase. To gain insight into the regulation of this promoter, we analyzed protein-DNA interactions by in vivo DMS and DNasel footprinting during the course of adenovirus infection. Little or no protein interaction at promoter sequences was detected early (5 hr) after infection but strong interactions at the major late transcription factor (MLTF/USF) binding site and at the TATA box were evident late (12 hr) after infection. Comparison of in vivo and in vitro footprints revealed that the in vivo interaction late after infection results from binding of the cellular transcription factor MLTF/USF. Nuclear extracts prepared from uninfected cells as well as cells harvested at 5 and 12 hr after infection contained similar levels of MLTF/USF footprint activity, therefore the lack of a detectable interaction early after infection is not due to reduced levels of the factor early in the viral growth cycle. Viral DNA replication was required for MLTF/USF binding at the major late promoter. These results indicate that DNA replication participates in the regulation of adenovirus late gene expression by facilitating the binding of a transcription factor to the major late promoter.
随着腺病毒感染从早期进入晚期，腺病毒主要晚期启动子的活性显著增加。为深入了解该启动子的调控机制，我们在腺病毒感染过程中通过体内二甲基亚砜（DMS）和DNA酶足迹法分析了蛋白质与DNA的相互作用。感染后早期（5小时），在启动子序列处几乎未检测到蛋白质相互作用，但在感染后期（12小时），在主要晚期转录因子（MLTF/USF）结合位点和TATA框处有明显的相互作用。体内和体外足迹的比较表明，感染后期的体内相互作用是由细胞转录因子MLTF/USF的结合引起的。从未感染细胞以及感染后5小时和12小时收获的细胞中制备的核提取物含有相似水平的MLTF/USF足迹活性，因此感染后早期未检测到相互作用并非由于病毒生长周期早期该因子水平降低所致。MLTF/USF在主要晚期启动子处的结合需要病毒DNA复制。这些结果表明，DNA复制通过促进转录因子与主要晚期启动子的结合参与腺病毒晚期基因表达的调控。
