TRANSLATION SYSTEM IN THE KINETOPLAST OF LEISHMANIA

所提出的研究的一个目标是验证存在一个功能 利什曼原虫动质体染色体的翻译系统 并研究其基本性质。这是必要的， 了解动质体在寄生虫生命周期中的功能。 翻译机器的组件的标识可以 提供了用于破坏生命周期的有用的治疗靶点， 治疗锥虫引起的疾病另外由于 翻译与RNA编辑产生的模板一起工作，这两个 系统可能以一种在其他真核生物中没有先例的方式相互作用， 系统.动质体核糖体与翻译调控的研究 因子可能揭示新的调节机制和结构 原则具体目标如下： #1.全基因组蛋白质的检测和表征 编辑的mRNA。这将用针对以下几种抗体进行研究： 在异源系统或合成系统中表达的线粒体蛋白 缩氨酸通过分析研究翻译水平的调控 在利什曼原虫的整个生命周期中，编辑的mRNA和蛋白质水平。 #2.动质体翻译系统的表征。到 研究从头蛋白质合成、放射性掺入 氨基酸转化为动质体中的高分子量产物， 线粒体将使用完整的细胞进行研究， 动质体-线粒体组分。重新开始模式的变化 将研究生命周期中合成的蛋白质。 #3.动质体核糖体的鉴定和分离。核糖体 将用9 S和12 S的特异性探针进行鉴定。rRNA和 核糖体蛋白S12的抗体。的功能评定 假定的核糖体将通过测定它们的翻译来进行 体内和体外感受态。我们将尝试重建 使用同源提取物的动质体翻译的无细胞系统 和纯化的核糖体。物理化学性质和组成 还将研究动基体核糖体。 #4. mRNA结合蛋白的鉴定和分离。为了 研究阻止翻译启动的机制， 不成熟的mRNA，特异性识别编辑的或预编辑的蛋白质， 将鉴定编辑的mRNA。

A goal of the proposed research is to verify existence of a functional system of translation in the kinetoplast-mitochondrion of Leishmania and investigate its basic properties. This is necessary for understanding the kinetoplast functions in the parasite's life cycle. Identification of the components of the translation machinery may provide useful therapeutical targets for disrupting the life cycle and treatment of trypanosomatid-caused diseases. In addition, since translation operates with templates produced by RNA editing, these two systems may interact in a way that has no precedent in other eukaryotic systems. Studies of kinetoplast ribosomes and translation regulatory factors may uncover novel regulatory mechanisms and structural principles. Specific aims are as follows: #1. Detection and characterization of proteins derived from fully edited mRNA. This will be investigated with antibodies against several mitochondrial proteins expressed in heterologous system or synthetic peptides. Regulation on translational level will be studied by analysis of edited mRNA and protein levels throughout the Leishmania life cycle. #2. Characterization of a translation system in the kinetoplast. To investigate de novo protein synthesis, incorporation of radioactive amino acids into high molecular weight products in kinetoplasts- mitochondria will be studied using intact cells and isolated kinetoplast-mitochondrial fraction. Changes in the pattern of de novo synthesized proteins during the life cycle will be investigated. #3. Identification and isolation of kinetoplast ribosomes. Ribosomes will be identified with specific probes for 9S and 12S. rRNA and antibodies against ribosomal protein S12. Functional evaluation of putative ribosomes will be performed by assaying their translation competence in vivo and in vitro. Attempts will be made to reconstitute a cell-free system of kinetoplast translation using homologous extract and purified ribosomes. Physico-chemical properties and composition of the kinetoplast ribosomes will also be studied. #4. Identification and isolation of mRNA binding proteins. In order to investigate mechanisms which prevent initiation of translation on immature mRNA, proteins which specifically recognize edited or pre- edited mRNA will be identified.