TRANSLATION SYSTEM IN THE KINETOPLAST OF LEISHMANIA

拟议研究的一个目标是验证函数的存在 利什曼原虫动质粒体内的翻译系统 并研究了它的基本性质。这是必要的，因为 了解该寄生虫生命周期中的动丝泡体功能。 翻译机器的部件的标识可以 为扰乱生命周期提供有用的治疗靶点 治疗锥虫引起的疾病。此外，由于 翻译使用由RNA编辑生成的模板进行操作，这两个模板 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。动粒体核糖体及其翻译调控的研究 因素可能揭示新的监管机制和结构性 原则。具体目标如下： #1.检测和表征完全来自于 编辑后的mRNA.这将用几种抗体进行调查 异源系统表达或人工合成的线粒体蛋白 多肽。对翻译层面的规范将通过分析进行研究 在利什曼原虫整个生命周期中编辑的信使核糖核酸和蛋白质水平。 #2.动质体内翻译系统的特征。至 研究从头蛋白质合成，放射性掺入 在动质体内转化为高相对分子质量的产物- 线粒体将使用完整的细胞和分离的细胞进行研究 动泡体--线粒体部分。从头开始的模式的变化 将对生命周期中合成的蛋白质进行研究。 3.动质体核糖体的鉴定和分离。核糖体 将被识别为9和12的特定探针。RRNA和 抗核糖体蛋白S12的抗体。功能评估 可能的核糖体将通过分析它们的翻译来执行 在体内和体外的能力。将尝试重建 一种使用同源提取物的无细胞动质外体翻译系统 和纯化的核糖体。黄曲霉毒素的理化性质和组成 动质体核糖体也将被研究。 4.mRNA结合蛋白的鉴定和分离。为了 调查阻止启动翻译的机制 未成熟的信使核糖核酸，是一种能识别编辑或预编码的蛋白质。 编辑后的信使核糖核酸将被识别。

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.