Marsh L E, Dreher T W, Hall T C
Department of Biology, Texas A&M University, College Station 77843-3258.
Nucleic Acids Res. 1988 Feb 11;16(3):981-95. doi: 10.1093/nar/16.3.981.
The subgenomic promoter of a (+)-stranded RNA virus, brome mosaic virus (BMV) controlling synthesis of subgenomic RNA4 has been defined in vitro. Truncated and mutant (-)-strand RNA templates were produced by in vitro transcription of cloned RNA3 cDNA. Subgenomic (+)-sense RNA was synthesized in vitro from these templates by a replicase (RNA-dependent RNA polymerase) preparation extracted from infected barley leaves. The activities of templates with truncations and deletions surrounding the RNA4 initiation site revealed a promoter of approximately 62 bases grouped into four functional domains. The core sequence consists of about twenty bases immediately upstream of, and including, the initiation nucleotide. In addition to the core sequence, a domain overlapping the 5' untranslated end of RNA4 apparently determines correct initiation. Two domains immediately upstream of the promoter core consist of the internal poly(A) tract of RNA3, which probably serves as an non base-paired spacer facilitating access of the replicase to the promoter, and a sequence, UUAUUAUU, that is required for high levels of promoter activity. Homologies to sequences surrounding the initiation sites of subgenomic RNAs from several plant RNA viruses, and from alphaviruses, have been detected.
已在体外确定了正链RNA病毒——雀麦花叶病毒（BMV）的亚基因组启动子，该启动子控制亚基因组RNA4的合成。通过对克隆的RNA3 cDNA进行体外转录，制备了截短和突变的负链RNA模板。利用从受感染的大麦叶片中提取的复制酶（RNA依赖性RNA聚合酶）制剂，从这些模板体外合成亚基因组正链RNA。对RNA4起始位点周围进行截短和缺失的模板活性分析表明，存在一个约62个碱基的启动子，该启动子可分为四个功能域。核心序列由起始核苷酸上游紧邻且包括起始核苷酸在内的约20个碱基组成。除核心序列外，一个与RNA4的5'非翻译末端重叠的结构域显然决定了正确的起始。启动子核心上游的两个结构域分别是RNA3的内部聚腺苷酸序列，其可能作为非碱基配对间隔区，便于复制酶接近启动子；还有一个序列UUAUUAUU，它是启动子高水平活性所必需的。已检测到该启动子与几种植物RNA病毒以及甲病毒亚基因组RNA起始位点周围序列的同源性。
