MOLECULAR BIOLOGY OF CHLOROPLAST RIBOSOMES

该项目将检验表达调节的假设 叶绿体核糖体蛋白基因的编码 烟草叶绿体 DNA 发生在 转录和翻译。 我们分析 rps12-rps7 转录本 处理并充分表征转剪接事件 发生在 rps12 mRNA 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 通过鉴定多肽编码核糖体蛋白 由克隆基因及其功能在体外合成 mRNA。 我们将评估转录的重要性 通过测量体内 mRNA 水平来调节这些基因 体外实验，检查 mRNA 的稳定性 几种叶绿体核糖体蛋白，评估 启动子序列转录调控，并探测 可能调节转录的蛋白质因子的证据 这些基因。 翻译控制机制将由以下人员评估 检查 mRNA 水平及其发生率的差异 含有内含子和缺乏核糖体内含子的翻译 蛋白质基因。 叶绿体核糖体基因是否翻译 通过核糖体蛋白的反馈抑制来调节 他们自己将受到考验。 核糖体结合位点效率 将比较其中的几个基因，并确定其重要性 通过位点特异性诱变检查的结合位点序列 实验后进行平移测量 体外效率。 rps12基因产物的功能将 进行检查，我们将尝试利用该基因来开发 用于叶绿体转化的选择标记。

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.