MOLECULAR BIOLOGY OF CHLOROPLAST RIBOSOMES

这个项目将检验这样一种假设，即对表达的调控 叶绿体核糖体蛋白基因的编码 烟草叶绿体DNA存在于 转录和翻译。我们分析了rps 12-rps 7转录本 处理并充分表征反式剪接事件 发生在rps12mRNA的成熟过程中。我们还将 建立叶绿体DNA的基因-基因产物关系 通过鉴定多肽来编码核糖体蛋白 是从克隆的基因和它们的功能 MRNAs。我们将评估转录的重要性 通过测量体内和外周血中的mRNA水平来调节这些基因 在体外实验中，检测信使核糖核酸的稳定性 几种叶绿体核糖体蛋白，评估了 转录调控的启动子序列，并探测 可能调节转录的蛋白质因子的证据 这些基因。翻译控制机制将通过以下方式进行评估 检测mRNA水平及其比率的差异 含内含子和缺内含子核糖体的翻译 蛋白质基因。叶绿体核糖体基因的翻译 是由核糖体蛋白的反馈抑制调节的 他们自己将受到考验。核糖体结合部位的效率 这些基因中的几个将被比较，并且其意义 用定点突变技术检测结合位点序列 随后的实验测量了平移 体外效率。Rps 12基因产物的功能将 我们将尝试利用这种基因来开发一种 叶绿体转化的可选择标记。

This project will test the hypothesis that regulation of expression of chloroplast ribosomal protein genes which are coded by Nicotiana tabacum chloroplast DNA occurs at the level of transcription and translation. We analyze rps12-rps7 transcript processing and fully characterize the trans-splicing event which takes place in the maturation of rps12 mRNA. We will also establish gene-gene product relationships for chloroplast DNA encoded ribosomal proteins by identifying the polypeptides which are synthesized in vitro from cloned genes and their functional mRNAs. We will assess the importance of transcriptional regulation of these genes by measuring mRNA levels in vivo and in vitro experiments, examine the stability of the mRNA for several chloroplast ribosomal proteins, assess the contribution of promoter sequences to transcriptional regulation, and probe for evidence of protein factors which might regulate transcription of these genes. Translational control mechanisms will be assessed by examining differences in mRNA levels and their rates of translation for intron-containing and intron lacking ribosomal protein genes. Whether translation of chloroplast ribosomal genes is regulated by a feedback inhibition by the ribosomal proteins themselves will be tested. Ribosome binding site efficiencies of several of these genes will be compared and the significance of the binding site sequences examined by site specific mutagenesis experiments followed by measurements of translational efficiencies in vitro. The function of the rps12 gene product will be examined and we will attempt to utilize this gene to develop a selectable marker for chloroplast transformation.