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

9630763 Maliga是高等植物的叶绿体基因组，编码的亚基与某些细菌DNA依赖的核糖核酸聚合酶亚基同源。该叶绿体RNAP所利用的启动子与大肠杆菌相似(70个启动子。叶绿体编码的RNAP启动子的选择依赖于核编码的(-样)因子。此外，一些启动子的转录活性受到核编码的转录因子与核心启动子上游元件相互作用的调节。一些报告表明，存在另一个定位于叶绿体的、核编码的RNAP。为了证实第二个叶绿体转录机制的存在，从烟草叶绿体基因组中删除了类大肠杆菌酶的必需亚单位的rpoB基因。在缺乏(亚单位((rpoB植物))的叶绿体中，转录本积累到相对较高的水平，编码基因表达装置的组成部分。因此，第二个RNA聚合酶被称为遗传系统(GS)RNAP。在(RpoB)叶绿体中，从启动子区域检测到转录，与(70个共有元件)没有相似性。本研究的目的是确定这两种RNA聚合酶在叶绿体功能和发育中的作用。该提案的重点是GS酶的作用。本研究的具体目标是：1)烟草叶绿体GS聚合酶启动子的鉴定。缺乏rpoA、rpoB、rpoC和rpoC2基因的烟草叶绿体中mRNAs丰富的基因将通过RNA凝胶印迹分析来鉴定。将为这些mRNA绘制转录起始点的图谱，并根据序列比较得出一致的序列。2)体内确定GS启动子的结构。通过检测突变启动子的报告基因在烟草叶绿体中的表达，将鉴定GS启动子的重要功能元件。3)GS启动子组织和细胞型特异性转录的测定。这将通过测试组织特异性的mRNAs积累和报告基因产物的免疫定位来实现。4)检测GS和类大肠杆菌RNAPs在叶绿体rRNA操纵子表达中的作用。RRNA操纵子含有GS和类大肠杆菌(70个启动子)。只有一个启动子的操纵子的表达将测试这些启动子中每个启动子在质体功能和发育中的作用。6)建立了GS转录机制的体外分析系统。将使用烟草提取物建立体外试验，在该试验中将鉴定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亚基基因的前提。植物含有被称为叶绿体的特殊细胞器，它能将光能转化为碳水化合物。与某些细菌系统相比，这些叶绿体有一种不寻常的基因表达装置。当这个装置被阻断时，可以看到另一个不同于细菌系统的基因表达系统。将对该系统进行研究，以确定它对植物叶和根组织中基因表达的贡献。***

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. ***