Structural Biology of Retrotransposition

逆转录转座的结构生物学

• 批准号: 10017691
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017691
• 关键词: Active Sites; Algae; Anacystisnidulans; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Cancer Etiology; Catalytic RNA; Complementary DNA; Complex; Cryoelectron Microscopy; DNA; Data; Development; Dissociation; Elements; Engineering; Escherichia coli; Eukaryota; Evolution; Exhibits; Foundations; Gene Expression; Genetic Structures; 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; thermostability; three dimensional structure; transcriptome

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 逆转位的结构生物学。非长末端重复序列(Non-LtR)逆转录元件是可移动的遗传 能够将自身复制并粘贴到DNA基因组中新位置的元件 转录酶和RNA中间体。这些反转录元件至少占人类基因组的46%。 关于它们整合成双链DNA的确切机制，人们知之甚少。线元素包括 人类基因组中一类特别丰富的逆转录元件，对基因有很大的影响 通过在转录启动子附近插入来表达。此外，异常的线状逆转位 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 经过30多年的研究，还没有一个原子模型可以解释逆转座的机制 分子水平。这是因为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的逆转录转座水平。第二类内含子被认为是 真核生物非LTR逆转录元件的祖先，如在人类中发现的LINE元件。第II组 由于其稳定性和高通量，逆转录元件是一种更易于处理的结构确定模型系统。 逆转录转座的体外活性。为了从结构上洞察逆转位，我们的目标是使用单一- 粒子冷冻-EM用于解决第二类内含子反转录元件的高分辨率结构。具体地说，我们将获得 逆转录转座到dsDNA不同阶段的逆转录元件的结构。我们将使用 生化、遗传和结构方法的组合：1)确定自由基团的结构 II内含子反转录元件。2)确定整合到靶双链DNA的机制。这是 预计与哺乳动物逆转录元件催化的逆转座有直接的相似之处。我们会 捕捉逆转录转座的不同阶段，创建整个过程的“分子电影”。这将是 代表了逆转座的第一个原子模型。