In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.
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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由核酮糖 1,5-二磷酸羧化酶大亚基基因编码的玉米叶绿体转录物的体外合成和加工。
DOI：
10.1128/mcb.5.10.2733-2745.1985
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发表时间：
1985
影响因子：
5.3
通讯作者：
Chua,NH
中科院分区：
文献类型：
--
作者：
Hanley-Bowdoin,L;OrozcoJr,EM;Chua,NH
利用玉米和豌豆叶绿体提取物和含有172碱基对（bp）和上游序列791 bp的克隆质体DNA模板，体外转录核酮糖1,5-二磷酸羧化酶大亚基基因（rbcL）。合成了三种主要的体外RNA，它们对应于体内的maizerbclrna，其5 '端分别位于蛋白质编码区上游300、100至105和63个核苷酸。在体外实验中，包括“- 300”5 ‘端（位于- 300位置的5 ’端）在内的109 bp的缺失抑制了allrbcl的转录。含有该109-bp片段的质粒DNA足以在体外指导正确的转录起始。克隆的模板含有191 bp的质体DNA，包括- 105和- 63rbcLtermini，在体外不支持转录。外源添加的−300 RNA可以通过玉米叶绿体提取物转化为−63转录物。这些结果表明- 300 RNA是主要的maizerbcl转录本，- 63 RNA是- 300转录本的加工形式，而- 105 RNA的合成依赖于- 300区域。maizerbcLgene的启动子位于−300位点两侧的109 bp内。在体外对- 300转录起始位点上游11 bp的109 bp叶绿体序列进行诱变，降低了drbcl启动子的活性。
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 maizerbcLprotein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maizerbcLRNAs 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 allrbcLtranscription 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 −63rbcLtermini, 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 maizerbcLtranscript, 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 maizerbcLgene 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 reducedrbcLpromoter activity in vitro.