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首页> 外文期刊>Molecular and Cellular Biology >In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.
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In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

机译：核糖1，5-双磷酸羧化酶大亚基基因编码的玉米叶绿体转录本的体外合成和加工。

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摘要

The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.
机译：使用玉米和豌豆叶绿体提取物和克隆的质体DNA模板体外转录核糖1,5-双磷酸羧化酶的大亚基基因（rbcL），该模板包含玉米rbcL蛋白编码区的172个碱基对（bp）和791 bp上游序列。合成了三种主要的体外RNA，分别对应于体内玉米rbcL RNA，其5'末端位于蛋白质编码区上游300、100至105和63个核苷酸处。 109 bp的缺失（包括“ -300” 5'端（位置-300处的5'端））在体外抑制了所有rbcL转录。含有该109 bp片段的质粒DNA足以指导体外正确的转录起始。克隆的模板包含191 bp的质体DNA，其中包括-105和-63 rbcL末端，不支持体外转录。玉米叶绿体提取物可将外源添加的-300 RNA转化为-63转录本。这些结果确定了-300 RNA是主要的玉米rbcL转录物，-63 RNA是-300转录物的加工形式，并且-105 RNA的合成取决于-300区域。玉米rbcL基因的启动子位于-300位点侧翼的109 bp内。突变的-300转录起始位点上游11 bp处的109 bp叶绿体序列降低了rbcL启动子活性。

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