ARCO Plant Cell Research Institute, 94566, Dublin, CA, USA.
Plant Mol Biol. 1983 Sep;2(5):279-90. doi: 10.1007/BF01578646.
Thein vitro DNA- or RNA-directed synthesis of the large subunit (LS) of spinach chloroplast ribulose-1,5-biphosphate carboxylase (RuP2C) has been examined in a highly definedE. coli transcription-translation system. Spinach chloroplast DNA, RNA and recombinant plasmids containing the spinach chloroplast LS gene (rbcL) have been used as templates in thein vitro system and a quantitative assay has been developed to measure LS formation. Thein vitro formed product contains formylmethionine at the N-terminal position and sediments primarily as a monomer. There is no detectable enzymatic activity associated with thein vitro product. To determine where theE. coli RNA polymerase used in these systems initiates, we have examined the transcripts produced by this enzymein vitro. Measurements of run-off transcripts indicate thatE. coli RNA polymerase initiates at the same position on the gene as is seenin vivo. In addition, the complete nucleotide sequence of therbcL gene including previously unsequenced 3' and 5' flanking regions has been determined. The sequence agrees, except at two nucleotide positions, with previously published sequencing data for this gene (Zurawski, G, Perrot, B, Bottomley, W, Whitfeld, PR, 1981. Nucleic Acids Res. 9:3251-3270).
已在高度定义的大肠杆菌转录-翻译系统中检查了菠菜叶绿体核酮糖-1,5-二磷酸羧化酶（RuP2C）大亚基（LS）的体外 DNA 或 RNA 指导合成。菠菜叶绿体 DNA、RNA 和含有菠菜叶绿体 LS 基因（rbcL）的重组质粒已被用作体外系统中的模板，并开发了一种定量测定法来测量 LS 的形成。体外形成的产物在 N 端位置含有甲硫氨酸，主要作为单体沉降。体外产物没有检测到相关的酶活性。为了确定这些系统中使用的大肠杆菌 RNA 聚合酶从何处开始，我们已经检查了该酶体外产生的转录物。运行转录物的测量表明，大肠杆菌 RNA 聚合酶在与体内相同的位置起始基因。此外，已确定了包括先前未测序的 3'和 5'侧翼区在内的完整 rbcL 基因的核苷酸序列。该序列除了在两个核苷酸位置外，与该基因先前发表的测序数据一致（Zurawski，G，Perrot，B，Bottomley，W，Whitfield，PR，1981。Nucleic Acids Res. 9:3251-3270）。
