RETROPLASMID AND GROUP II INTRON REVERSE TRANSCRIPTASES

拟议的研究包括持续的生化遗传学研究。 线粒体[质粒和第二组内含子，两种逆转录元件 在细胞器和细菌中发现的物质。这些逆行元素 编码独特物种的逆转录酶(RTS)并使用新的 复制和移动机制，可提供对 逆转录元件和内含子的早期进化。莫里克维尔和瓦尔库德 某些脉孢子菌中发现的线粒体反转录质粒(MRPs) 是密切相关的小环状DNA，通过反向复制 抄写。在目前的授权期内，我们发现MRP- 编码的RT的功能类似于病毒依赖的RNA聚合酶 在启动在tRNA样结构上的(-)链cDNA合成中 3‘端的MRP转录本，并具有前所未有的DNA能力 聚合酶启动DNA从头合成(即，没有引子)。 这些特征是从原始RT演变而来的 一种依赖于RNA的RNA聚合酶，他们支持这样的假设 MRP是与人类早期祖先有关的原始逆转录元素 逆转录病毒。在建议的研究中，我们会继续研究 MRP、其RT及其复制和整合机制。集团化 Ii内含子是拟议研究的第二个主题，具有催化作用。 被认为与核前mRNA的前体有关的RNA 内含子。酵母线粒体DNA第二组内含子COX1-I1和-I2(AI1和AI2) 已经被证明编码类似RT的蛋白质，这种蛋白质在拼接 编码它们的内含子。与菲利普博士合作 Perlman和Ronald Butow(美国德克萨斯州)，我们展示了AI1和AI2 蛋白质也是活性的、内含子特有的转录因子，在内含子中发挥作用 机动性。在建议的研究中，我们会继续研究 第二类内含子RTS在内含子移动和RNA剪接中发挥作用。 对MRP和第二类内含子的持续研究应该会提供新的 对逆转录和复制机制的洞察，结构性 以及不同类型的核酸之间的进化关系 聚合酶、逆转录元件和RNA病毒群的进化 其中包括艾滋病毒和其他重要的人类病原体。具体目标是： (1)继续进行MRP RT的生化分析，重点是De Novo引发反应与模板和底物的作用方式 承认。(2)研究MRP复制途径的其他方面， 包括RT翻译、()链合成、DNA环化和 整合。(3)建立MRP RT的表达方法 结构功能分析。(4)继续开展协同实验 与菲利普·帕尔曼博士和罗纳德·布托博士一起研究 酵母tDNA组II内含子RTS在内含子移动和RNA剪接中的作用。(5) 发展细菌或其他第II类基因的表达方法 大肠杆菌中的内含子RTS。

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.