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IN VITRO TRANSCRIPTION OF CHLOROPLAST DNA GENES

叶绿体 DNA 基因的体外转录

基本信息

批准号：
3279528
负责人：
KRISHNA K TEWARI
金额：
$ 18.5万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-03-01 至 1990-03-31
项目状态：
已结题

项目摘要

The long term objective is to study the mechanism of transcription of chloroplast DNA genes using homologous RNA polymerase. We have purified an RNA polymerase preparation from pea chloroplasts that shows strong preference to chloroplast DNA templates and can synthesize in vitro full length rRNA, tRNA, and photogene 32 mRNA transcripts using cloned genes. We now propose to study the following: 1) A detailed study on the nucleotide sequences required (promoter sequences) for recognition by the RNA polymerase for all three types of RNA chains i.e. rRNA, tRNA, and mRNA. These experiments will be carried out by constructing deletion mutants of the sequences that surround transcription initiation sites of genes using Ba131. Identification of the sequences at the 5' end of genes required for the transcription will be followed by experiments where single and multiple point mutations within the control region will be produced and the mutated DNA studied for transcription. These experiments will enable us to precisely identify the nucleotides that constitute specific promoters of each type of gene; 2) An analysis of nucleotide sequences at the 3' end of the genes that might be involved in the temination of transcription; 3) Further purification of the RNA polymerase enzyme using conventional purification procedures to establish whether there is a single RNA polymerase that transcribes different types of chloroplast DNA genes or there are multiple polymerases each specific for ribosomal RNA, transfer RNA, and messenger RNA genes; and 4) Analyze the structure of RNA polymerase(s) using monoclonal antibodies. These studies will, in future, be followed by experiments that would identify polypeptides which control the initiation, elongation and termination of chloroplast RNA transcripts.

长期的目标是研究转录的机制，叶绿体DNA基因的同源RNA聚合酶。我们净化了一个来自豌豆叶绿体的RNA聚合酶制剂，优先选择叶绿体DNA模板并可体外完整合成长度rRNA、tRNA和photogene 32 mRNA转录本。我们现建议进行以下研究：（一）详细研究识别所需的核苷酸序列（启动子序列）所有三种类型的RNA链，即rRNA、tRNA和 mRNA。这些实验将通过构建缺失转录起始位点周围序列的突变体使用Ba 131的基因。基因5'端序列的鉴定转录所需的将遵循实验，其中单一并且将在控制区内产生多个点突变，突变的DNA进行转录研究。这些实验将使我们可以精确地识别构成特定启动子的核苷酸 2）3'端核苷酸序列分析可能参与转录终止的基因; 3） RNA聚合酶的进一步纯化使用常规的纯化方法。纯化程序，以确定是否存在单个RNA 转录不同类型叶绿体DNA基因的聚合酶，或存在多种聚合酶，每种聚合酶对核糖体RNA、转移 RNA和信使RNA基因;和4）分析RNA的结构使用单克隆抗体的聚合酶。这些研究将在未来，随后进行实验，以鉴定控制叶绿体RNA转录物的起始、延伸和终止。