RETROPLASMID AND GROUP II INTRON REVERSE TRANSCRIPTASES

逆质体和 II 组内含子逆转录酶

• 批准号: 2179057
• 负责人: ALAN M. LAMBOWITZ
• 金额: $ 31.79万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179057
• 关键词: DNA replication; Neurospora; RNA directed DNA polymerase; RNA splicing; complementary DNA; enzyme mechanism; enzyme structure; evolution; fungal genetics; gene expression; laboratory rabbit; mitochondria; mitochondrial DNA; molecular cloning; nucleic acid sequence; nucleic acid structure; plasmids; polymerase chain reaction; protein sequence; protein structure function; ribonucleoproteins; ribozymes; site directed mutagenesis; transfer RNA; transposon /insertion element

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.

拟议的研究涉及持续的生化遗传学研究 线粒体[质粒和第二组内含子，两种类型的逆转录元件 已在细胞器和细菌中发现。这些复古元素 编码独特种类的逆转录酶 (RT) 并使用新颖的 复制和移动机制可以提供对 逆转录因子和内含子的早期进化。莫里斯维尔和瓦尔库德 在某些脉孢菌菌株中发现线粒体逆转录质粒 (mRP) 是密切相关的小环状DNA，通过反向复制 转录。在当前的资助期内，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 tRNA 样结构处启动 (-) 链 cDNA 合成 mRP 转录本的 3' 末端，具有前所未有的 DNA 能力 聚合酶启动 DNA 从头合成（即无需引物）。 这些特征是由原始 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 是与早期祖先相关的原始逆转录因子 逆转录病毒。在拟议的研究中，我们将继续研究 mRP、它们的 RT 以及它们的复制和整合机制。团体 II 内含子是拟议研究的第二个主题，具有催化作用 RNA被认为与核前mRNA的祖细胞有关 内含子。酵母 mtDNA II 组内含子 COX1-I1 和 - I2（aI1 和 aI2） 已被证明编码类似 RT 的蛋白质，其功能是剪接 它们被编码的内含子。与博士合作。菲利普 Perlman 和 Ronald Butow（德克萨斯州），我们证明了 aI1 和 aI2 蛋白质也是活跃的、内含子特异性 RT，在内含子中发挥作用 流动性。在拟议的研究中，我们将继续研究如何 II 组内含子 RT 在内含子移动性和 RNA 剪接方面发挥作用。 mRP 和 II 组内含子的持续研究应该提供新的 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、逆转录元件和RNA病毒、组的进化 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mRP RT的生化分析，重点关注de novo引发反应以及模板和引物的模式 认出。 (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 一体化。 (3) 开发mRP RT的表达方法 结构功能分析。 （四）继续合作实验 与博士。菲利普·帕尔曼和罗纳德·布图研究了 酵母 tDNA II 组内含子 RT 在内含子移动性和 RNA 剪接中。 (5) 开发细菌或其他 II 类细菌的表达方法 大肠杆菌中的内含子 RT。