RETROPLASMID AND GROUP II INTRON REVERSE TRANSCRIPTASES

拟议的研究涉及持续的生物化学遗传学研究， 线粒体[质粒和II组内含子，两种类型的逆转录元件 在细胞器和细菌中发现的。这些复古元素 编码独特种类的逆转录酶（RT）并使用新的 复制和移动机制，可以提供深入了解 反转录元件和内含子的早期进化。莫里斯维尔和瓦库德 在某些脉孢菌菌株中发现的线粒体逆转录质粒（mRPs） 是紧密相关的小环状DNA， 转录。在目前的补助金期间，我们发现，mRP- 编码的RT功能类似于病毒RNA依赖性RNA聚合酶 在起始（-）链cDNA合成中， mRP转录本的3'末端，并具有前所未有的DNA 聚合酶以启动DNA从头合成（即，没有底漆）。 这些特征被认为是原始RT的特征， 一种依赖RNA的RNA聚合酶，他们支持这一假设， mRPs是与早期祖先相关的原始逆转录因子， 逆转录病毒在建议的研究中，我们会继续研究 mRPs，它们的RT和它们的复制和整合机制。组 第二个研究对象是内含子，它具有催化作用 认为与核前mRNA祖细胞相关的RNA 内含子酵母线粒体DNA第二组内含子COX 1-I1和- I2（aI 1和aI 2） 已被证明编码RT样蛋白，其功能是剪接 它们被编码的内含子。与菲利普博士合作 帕尔曼和罗纳德布托（u。我们发现aI 1和aI 2 蛋白质也是活性的，内含子特异性的RT，其在内含子中起作用。 迁移率在建议的研究中，我们会继续研究 II组内含子RT在内含子迁移和RNA剪接中起作用。 对mRPs和第二组内含子的继续研究将提供新的 深入了解逆转录和复制机制，结构 以及不同类型核酸之间的进化关系 聚合酶，逆转录因子和RNA病毒的进化， 包括艾滋病病毒和其他重要的人类病原体。具体目标是： (1)为了继续对mRP RT进行生化分析，重点是检测 从头引发反应及模板和引物的模式 识别. (2)为了研究mRP复制途径的其他方面， 包括RT翻译、（+）链合成、DNA环化和 一体化(3)为了开发表达mRP RT的方法， 结构功能分析(4)继续合作实验 菲利普·帕尔曼和罗纳德·布托博士一起研究了 酵母tDNAII组内含子RTs在内含子迁移率和RNA剪接中的作用。（五） 开发用于表达细菌或其他组II的方法， E.杆菌

The proposed research involves continued biochemical-genetic studies of mitochondrial [plasmids and group II introns, two types of retroelements that have been found in organelles and bacteria. These retroelements encode unique species of reverse transcriptases (RTs) and use novel replication and mobility mechanisms that may provide insight into the early evolution of retroelements and introns. The Mauriceville and Varkud mitochondrial retroplasmids (mRPs) found in certain strains of Neurospora are closely related, small circular DNAs that replicate via reverse transcription. During the current grant period, we found that the mRP- encoded RT functions analogously to viral RNA-dependent RNA polymerases in initiating (-) strand cDNA synthesis at a tRNA-like structure at the 3' end of the mRP transcript and has the unprecedented ability for a DNA polymerase to initiate DNA synthesis de novo (i.e., without a primer). These characteristics are expected for a primitive RT that evolved from an RNA-dependent RNA polymerase, and they support the hypothesis that the mRPs are primitive retroelements related to the early ancestors of retroviruses. In the proposed research, we would continue to study the mRPs, their RT and their mechanisms of replication and integration. Group II introns, the second subject of the proposed research, are catalytic RNAs believed to be related to the progenitors of nuclear pre-mRNA introns. The yeast mtDNA group II introns COX1-I1 and - I2 (aI1 and aI2) had been shown to encode RT-like proteins that function in splicing the intron in which they are encoded. In collaboration with Drs. Philip Perlman and Ronald Butow (u. Texas), we showed that the aI1 and aI2 proteins are also active, intron-specific RTs that function in intron mobility. In the proposed research, we would continue to study how the group II intron RTs function in both intron mobility and RNA splicing. The continued studies of mRPs and group II introns should provide novel insight into reverse transcription and replication mechanisms, structural and evolutionary relationships between different types of nucleic acid polymerases, and the evolution of retroelements and RNA viruses, groups that include HIV and other important human pathogens. Specific aims are: (1) To continue biochemical analysis of the mRP RT, focusing on the de novo initiation reaction and the modes of template and primer recognition. (2) To study other aspects of the mRP replication pathway, including RT translation, (+) strand synthesis, DNA circularization and integration. (3) To develop methods for expression of the mRP RT for structure-function analysis. (4) To continue collaborative experiments with Drs. Philip Perlman and Ronald Butow to study the function of the yeast tDNA group II intron RTs in intron mobility and RNA splicing. (5) To develop methods for the expression of bacterial or other group II intron RTs in E. coli.