Omilli F, Ernoult-Lange M, Borde J, May E
Mol Cell Biol. 1986 Jun;6(6):1875-85. doi: 10.1128/mcb.6.6.1875-1885.1986.
We analyzed the sequences involved in vivo in the initiation of simian virus 40 (SV40) late transcription occurring in the absence of both SV40 origin sequences and T antigen. The constituent elements of the SV40 late promoters have already been the subject of extensive studies. In vitro studies have resulted in the description of two putative domains of the late promoters. The first domain consists of an 11-base-pair (bp) sequence, 5'-GGTACCTAACC-3', located 25 nucleotides (nt) upstream of the SV40 major late initiation site (MLIS) (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:624-633, 1982). The second domain is located within the G-C-rich region (J. Brady, M. Radonovich, M. Thoren, G. Das, and N. P. Salzman, Mol. Cell. Biol. 4:133-141; U. Hansen and P. A. Sharp, EMBO J. 2:2293-2303). Our previous in vivo studies permitted us to define a domain of the late promoter which extends from nt 332 to nt 113 and includes the 72-bp enhancer sequences. Here, by using transfection of the appropriate chimeric plasmids into HeLa cells in conjunction with quantitative S1 nuclease analysis, we analyzed in more detail the sequences required for the control of SV40 late-gene expression occurring before the onset of viral DNA replication. We showed that the major late promoter element is in fact the 72-bp repeat enhancer element. This element was able to drive efficient late transcription in the absence of T antigen. Under our experimental conditions, removal of the G-C-rich region (21-bp repeats) entailed a significant increase in the level of late-gene expression. Moreover, translocation of this element closer to the MLIS (53 nt upstream of the MLIS) enhanced the level of transcripts initiated at natural late initiation sites. Our results suggest that the G-C-rich regions have to be positioned between the enhancer element and the initiation sites to stimulate transcription from downstream sites. Thus, the relative arrangement of the various promoter elements is a critical factor contributing to the situation in which the early promoter is stronger than late promoters before viral DNA replication.
我们分析了在没有猿猴病毒40（SV40）起始序列和T抗原的情况下，体内发生的SV40晚期转录起始所涉及的序列。SV40晚期启动子的组成元件已经是广泛研究的对象。体外研究已经描述了晚期启动子的两个假定结构域。第一个结构域由一个11个碱基对（bp）的序列5'-GGTACCTAACC-3'组成，位于SV40主要晚期起始位点（MLIS）上游25个核苷酸（nt）处（J. Brady、M. Radonovich、M. Vodkin、V. Natarajan、M. Thoren、G. Das、J. Janik和N. P. Salzman，《细胞》31:624 - 633，1982）。第二个结构域位于富含G - C的区域内（J. Brady、M. Radonovich、M. Thoren、G. Das和N. P. Salzman，《分子与细胞生物学》4:133 - 141；U. Hansen和P. A. Sharp，《欧洲分子生物学组织杂志》2:2293 - 2303）。我们之前的体内研究使我们能够确定晚期启动子的一个结构域，该结构域从nt 332延伸到nt 113，包括72bp的增强子序列。在这里，通过将适当的嵌合质粒转染到HeLa细胞中并结合定量S1核酸酶分析，我们更详细地分析了在病毒DNA复制开始之前控制SV40晚期基因表达所需的序列。我们表明，主要的晚期启动子元件实际上是72bp的重复增强子元件。该元件能够在没有T抗原的情况下驱动高效的晚期转录。在我们的实验条件下，去除富含G - C的区域（21bp重复序列）导致晚期基因表达水平显著增加。此外，将该元件向MLIS靠近（MLIS上游53nt）会增强在天然晚期起始位点起始的转录本水平。我们的结果表明，富含G - C的区域必须位于增强子元件和起始位点之间，以刺激下游位点的转录。因此，各种启动子元件的相对排列是导致在病毒DNA复制之前早期启动子比晚期启动子更强这种情况的一个关键因素。
