The Role of a Nuclear-Encoded Plastid RNA Polymerase in Plastid Function and Development

核编码质体 RNA 聚合酶在质体功能和发育中的作用

• 批准号: 9630763
• 负责人: Pal Maliga
• 金额: $ 28.5万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9630763&HistoricalAwards=false
• 关键词: Role; Nuclear; Encoded; Plastid; RNA

9630763 Maliga The plastid genome of higher plants encodes subunits which are homologous to certain bacterial DNA-dependent RNA polymerase (RNAP) subunits. The promoters utilized by this plastid RNAP are similar to E. coli (70 promoters. Promoter selection by the plastid-encoded RNAP is dependent on nuclear-encoded (-like factors. In addition, transcription activity from some promoters is modulated by nuclear-encoded transcription factors interacting with elements upstream of the core promoter. Several reports have suggested the existence of an additional plastid-localized, nuclear-encoded RNAP. To confirm the existence of a second plastid transcription machinery, the rpoB gene for the essential ( subunit of the E. coli-like enzyme was deleted from the tobacco plastid genome. Transcripts in plastids lacking the ( subunit ((rpoB plants) accumulate to relatively high levels for genes which encode components of the gene expression apparatus. Therefore, the second RNA polymerase is referred to as the genetic system (GS) RNAP. In the (rpoB plastids transcription was detected from promoter regions with no similarity to (70 consensus elements. The research objective is to identify the role of the two RNA polymerases in plastid function and development. The proposal is focused on the role of the GS enzyme. The specific objectives of the research are: 1) Identification of promoters for the GS polymerase in tobacco plastids. Genes for which mRNAs are abundant in tobacco plastids lacking the rpoA, rpoB and rpoCl and rpoC2 genes will be identified by RNA gel blot analysis. The transcription initiation sites will be mapped for these mRNAs, and a consensus sequence will be derived based on sequence comparison. 2) In vivo definition of GS promoter architecture. Functionally important elements of GS promoters will be identified by testing expression of reporter genes from the mutant promoters in vivo in tobacco plastids. 3) Determination of tissue and cell-type specific transcription from GS pr omoters. This will be accomplished by testing tissue-specific accumulation of mRNAs and immunolocalization of reporter gene products. 4) Testing the role of GS and E. coli-like RNAPs in the expression of the plastid rRNA operon. The rRNA operon has both GS and E. coli-like (70 promoters. Expression of the operon from only one promoter will test role of each of these promoters in plastid function and development. 6) Developing an in vitro system for the analysis of the GS transcription machinery. An in vitro assay will be established using tobacco extracts in which GS RNAP transcriptional properties and GS promoter functional elements will be identified. Purification of the GS RNAP will facilitate the identification of subunit composition, the prerequisite of cloning GS subunit genes. %%% Plants contain specialized organelles called chloroplasts which convert light energy to carbohydrates. These chloroplasts have an unusual gene expression apparatus which compares with certain bacterial systems. When this apparatus is blocked another gene expression system is seen that is unlike the bacterial system. This system will be studied to determine what contribution it makes to plant gene expression in leaf and root tissues. ***

高等植物的质体基因组编码与某些细菌DNA依赖性RNA聚合酶（RNAP）亚基同源的亚基。 该质体RNAP利用的启动子与E.大肠杆菌（70个启动子。 质体编码的RNAP对启动子的选择依赖于核编码的类β因子。 此外，来自一些启动子的转录活性由与核心启动子上游元件相互作用的核编码转录因子调节。 一些报道表明存在另外的质体定位的、核编码的RNAP。 为了证实第二个质体转录机制的存在，将E.类大肠杆菌酶从烟草质体基因组中缺失。 在缺乏（rpoB植物）亚基的质体中转录物积累到相对高水平的编码基因表达装置组分的基因。 因此，第二RNA聚合酶被称为遗传系统（GS）RNAP。 在（rpoB）质体中，从与（70）共有元件不相似的启动子区域检测到转录。 研究目的是确定这两种RNA聚合酶在质体功能和发育中的作用。 该提案的重点是GS酶的作用。 本研究的具体目的是：1）烟草质体GS聚合酶启动子的鉴定。 将通过RNA凝胶印迹分析鉴定在缺乏rpoA、rpoB和rpoCl和rpoC 2基因的烟草质体中mRNA丰富的基因。 将绘制这些mRNA的转录起始位点，并基于序列比较推导出共有序列。 2)GS启动子结构的体内定义。 GS启动子的功能重要元件将通过测试报告基因在烟草质体中的体内突变启动子的表达来鉴定。3)从GS启动子确定组织和细胞类型特异性转录。 这将通过检测mRNA的组织特异性积累和报告基因产物的免疫定位来实现。 4)测试GS和E的作用。在质体rRNA操纵子的表达中的大肠杆菌样RNAP。 rRNA操纵子具有GS和E。coli-like（70个启动子。仅从一个启动子表达操纵子将测试这些启动子中的每一个在质体功能和发育中的作用。 6)建立了一个体外系统，用于分析GS转录机制。 将使用烟草提取物建立体外测定，其中将鉴定GS RNAP转录特性和GS启动子功能元件。GS RNAP的纯化将有助于亚基组成的鉴定，这是克隆GS亚基基因的先决条件。 植物中有一种特殊的细胞器叫做叶绿体，它能将光能转化为碳水化合物。 与某些细菌系统相比，这些叶绿体具有不寻常的基因表达装置。 当这个装置被阻断时，就会看到另一个与细菌系统不同的基因表达系统。 该系统将被研究，以确定它对植物基因在叶和根组织中表达的贡献。 ***