Structural Biology of Retrotransposition

逆转录转座的结构生物学

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要逆转录转座的结构生物学。非长末端重复（非 LTR）逆转录元件是可移动遗传的能够使用反向复制和粘贴自身到 DNA 基因组中新位置的元素转录酶和RNA中间体。这些逆转录元件至少占人类基因组的 46% 人们对它们整合到双链 DNA 中的精确机制知之甚少。线元素是人类基因组中一类特别丰富的逆转录因子，对基因有很大影响通过在转录启动子附近插入来表达。此外，LINE 的异常逆转录转座元素可通过破坏肿瘤抑制基因而导致癌症的发生。尽管更多经过30多年的研究，还没有一个原子模型能够解释逆转录转座的机制。分子水平。这是因为 LINE 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转座。 II 组内含子被认为是真核非 LTR 逆转录元件的祖先，例如人类中发现的 LINE 元件。第二组由于其稳定性和高通量，逆转录元件是一种更容易处理的结构测定模型系统。体外逆转录转座活性。为了深入了解逆转录转座的结构，我们的目标是使用单粒子冷冻电镜可解析 II 族内含子逆转录元件的高分辨率结构。具体来说，我们将获得逆转录转座到双链DNA的不同阶段期间逆转录元件的结构。我们将使用一个结合生化、遗传和结构方法：1) 确定自由基团的结构 II 内含子逆转录因子。 2) 确定整合到目标双链DNA中的机制。这是预计与哺乳动物逆转录因子催化的逆转录转座有直接相似之处。我们将捕捉逆转录转座的不同阶段，创建整个过程的“分子电影”。这将代表了逆转录转座的第一个原子模型。