Schweizer P, Mösinger E
Sandoz Agro Ltd., Agrobiological Research Station, Witterswil, Switzerland.
Plant Mol Biol. 1994 Apr;25(1):115-30. doi: 10.1007/BF00024203.
The development of plant in vitro transcription systems transcribing faithfully and efficiently from a broad range of plant nuclear promoters has remained a challenge. We examined the nucleotide sequence requirements for faithful and efficient transcription in a wheat germ chromatin extract (Yamazaki et al., Plant Mol Biol Rep 8: 114-123). The wheat germ chromatin extract was tested with a series of chimeric promoter constructs containing plant promoter sequences upstream from the TATA box, TATA boxes, and cap-site sequences (from -10 to +14, relative to the major in vivo initiation site) in different combinations. The plant extract transcribed faithfully from several chimeric promoters containing the capsite sequence of the parsley chalcone synthase promoter. The transcription was sensitive to the RNA polymerase II-specific inhibitor alpha-amanitin and was only dependent on the chalcone synthase cap-site sequence which therefore fulfils the operational criteria for a plant initiator element. Mutations of the putative chalcone synthase initiator element defined a core sequence '5'TAACAAC' around the initiation site that was necessary for efficient transcription in vitro. In contrast to the extract, purified wheat germ RNA polymerase II showed no preference for transcription from the major chalcone synthase in vivo initiation site.
开发能从多种植物核启动子进行准确高效转录的植物体外转录系统一直是一项挑战。我们研究了小麦胚染色质提取物中准确高效转录所需的核苷酸序列要求（Yamazaki等人，《植物分子生物学报告》8：114 - 123）。用一系列嵌合启动子构建体对小麦胚染色质提取物进行测试，这些构建体包含位于TATA框上游的植物启动子序列、TATA框以及不同组合的帽位点序列（相对于主要体内起始位点，从 - 10到 + 14）。植物提取物能从几个含有欧芹查尔酮合酶启动子帽位点序列的嵌合启动子进行准确转录。转录对RNA聚合酶II特异性抑制剂α - 鹅膏蕈碱敏感，并且仅依赖于查尔酮合酶帽位点序列，因此该序列符合植物起始元件的操作标准。推测的查尔酮合酶起始元件的突变确定了起始位点周围的核心序列“5'TAACAAC”，这是体外高效转录所必需的。与提取物不同，纯化的小麦胚RNA聚合酶II对从主要查尔酮合酶体内起始位点的转录没有偏好。
