Structural Biology of Retrotransposition

逆转录转座的结构生物学

• 批准号: 9290480
• 负责人: Navtej Singh Toor
• 金额: $ 30.46万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9290480
• 关键词: Active Sites; Algae; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Cancer Etiology; Catalytic RNA; Complementary DNA; Complex; DNA; Data; Development; Dissociation; Elements; Engineering; Escherichia coli; Eukaryota; Evolution; Exhibits; Foundations; Gene Expression; Genetic Transcription; Genome; Goals; Human; Human Genome; In Vitro; Introns; Invaded; Knowledge; Location; Malignant Neoplasms; Mobile Genetic Elements; Modeling; Molecular; Molecular Genetics; Molecular Weight; Monitor; Mutagenesis; Paste substance; Phylogenetic Analysis; Plasmids; Process; Proteins; RNA; RNA Binding; RNA Probes; RNA-Directed DNA Polymerase; Reaction; Resolution; Retroelements; Retrotransposition; Ribonucleoproteins; Role; Sequence Homology; Structure; Tumor Suppressor Genes; density; ds-DNA; genetic approach; improved; in vivo; insight; mammalian genome; movie; mutant; particle; promoter; reconstitution; structural biology; therapeutic development; therapy development; thermostability; three dimensional structure

PROJECT SUMMARY/ABSTRACT Structural biology of retrotransposition. Non-long terminal repeat (non-LTR) retroelements are mobile genetic elements that are able to copy and paste themselves into new locations in DNA genomes using a reverse transcriptase and an RNA intermediate. These retroelements comprise at least 46% of the human genome and little is known about their precise mechanism of integration into double stranded DNA. LINE elements are a particularly abundant class of retroelements in the human genome and have large effects on gene expression through insertion near transcription promoters. In addition, aberrant retrotransposition by LINE elements can result in the development of cancer through disruption of tumor suppressor genes. Despite more than 30 years of study, there is no atomic model that would explain the mechanism of retrotransposition at the molecular level. This is due to the fact that LINE elements are difficult targets for structure determination because they exhibit very low levels of retrotransposition in vitro. Group II introns are thought to be the ancestors of eukaryotic non-LTR retroelements, such as the LINE elements found in humans. Group II retroelements are a more tractable model system for structure determination due to their stability and high activity of retrotransposition in vitro. To gain structural insight into retrotransposition, we aim to use single- particle cryo-EM to solve high-resolution structures of a group II intron retroelement. Specifically, we will obtain structures of the retroelement during the different stages of retrotransposition into dsDNA. We will use a combination of biochemical, genetic, and structural approaches to: 1) Determine the structure of a free group II intron retroelement. 2) Determine the mechanism of integration into a target double-stranded DNA. This is expected to have direct parallels with retrotransposition catalyzed by mammalian retroelements. We will capture different stages of retrotransposition to create a `molecular movie' of the entire process. This will represent the first atomic model of retrotransposition.

项目摘要/摘要 逆转录的结构生物学。非长末端重复（非LTR）恢复元素是移动遗传 能够使用反向将自己复制并粘贴到DNA基因组中的新位置的元素 转录酶和RNA中间体。这些追溯元素至少占人类基因组的46％ 对于它们的精确整合到双链DNA中的机制知之甚少。线元素是 人类基因组中特别丰富的追溯元素类别，对基因具有很大的影响 通过插入附近的转录启动子表达。此外，逐线逆转逆转 元素可以通过破坏肿瘤抑制基因而导致癌症的发展。尽管有更多 超过30年的研究，没有原子模型可以解释在 分子水平。这是由于线元素是结构确定的困难目标 因为它们在体外表现出非常低水平的逆转录置。第二组内含子被认为是 真核非LTR恢复元素的祖先，例如人类中发现的线元素。第二组 重新元素是一个更可访问的模型系统，用于确定结构，并且由于其稳定性高 体外逆转录的活性。为了获得对逆转录的结构见解，我们的目标是使用单一 粒子冷冻EM求解II组内含子元素的高分辨率结构。具体来说，我们将获得 逆转录置置分为dsDNA的不同阶段的恢复元素的结构。我们将使用一个 生化，遗传和结构方法的结合：1）确定自由组的结构 II内含子恢复。 2）确定整合到目标双链DNA中的机制。这是 预计将与哺乳动物逆转录元素催化的逆转录座有直接相似之处。我们将 捕获反转词的不同阶段，以创建整个过程的“分子电影”。这会 代表了逆转录置位的第一个原子模型。