Fenoll C, Schwarz J J, Black D M, Schneider M, Howell S H
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853-1801.
Plant Mol Biol. 1990 Dec;15(6):865-77. doi: 10.1007/BF00039426.
Maize streak virus (MSV) is transcribed bidirectionally from an intergenic region and rightward transcription produces an RNA that encodes the coat protein. The intergenic region contains promoter elements required for rightward transcription including an upstream activating sequence (UAS) which endows the promoter with full activity in a maize transient expression system. The UAS contains two GC-rich repeats (GC boxes) and a long inverted repeat or hairpin with a loop harboring a TAATATTAC sequence common to all geminiviruses. Deletions through the UAS demonstrated the presence of an element, called the rightward promoter element (rpe1), which is responsible for transcriptional activation. Rpe1 includes the two GC-rich boxes, which are similar in sequence to Sp1 binding sites in mammalian cells, but not the conserved hairpin loop. Rpe1 binds maize nuclear factors in vitro and the characteristics of the binding interaction have been determined by 1) binding competition with oligonucleotides, 2) methidiumpropyl-EDTA footprinting and 3) methylation interference assays. Binding of maize nuclear factors to the UAS generates two major bands, slow and fast migrating bands, in gel retardation assays. Footprinting and factor titration data suggest that the fast bands arise by the binding of factors to one GC box while the slow bands are generated by factors binding to both boxes. The data further indicate that the factors bind to the two GC-rich boxes with little cooperativity and bind on opposite faces of the DNA helix.
玉米条纹病毒(MSV)从基因间隔区双向转录,向右转录产生一种编码外壳蛋白的RNA。基因间隔区包含向右转录所需的启动子元件,包括一个上游激活序列(UAS),该序列赋予启动子在玉米瞬时表达系统中的完全活性。UAS包含两个富含GC的重复序列(GC盒)和一个长的反向重复序列或发夹结构,其环中含有所有双生病毒共有的TAATATTAC序列。通过UAS的缺失证明了一种称为向右启动子元件(rpe1)的元件的存在,它负责转录激活。Rpe1包括两个富含GC的盒,其序列与哺乳动物细胞中的Sp1结合位点相似,但不包括保守的发夹环。Rpe1在体外与玉米核因子结合,并且通过以下方法确定了结合相互作用的特征:1)与寡核苷酸的结合竞争;2)甲硫基丙基-EDTA足迹分析;3)甲基化干扰分析。在凝胶阻滞分析中,玉米核因子与UAS的结合产生两条主要条带,即慢迁移条带和快迁移条带。足迹分析和因子滴定数据表明,快迁移条带是由因子与一个GC盒结合产生的,而慢迁移条带是由因子与两个盒结合产生的。数据进一步表明,这些因子以很少的协同性与两个富含GC的盒结合,并结合在DNA螺旋的相对面上。