Structural Biology of Retrotransposition

项目总结/摘要 反转录转座的结构生物学非长末端重复序列（non-LTR）逆转录元件是移动的遗传的 能够复制和粘贴到DNA基因组中的新位置的元素， 转录酶和RNA中间体。这些逆转录因子至少占人类基因组的46% 并且关于它们整合到双链DNA中的精确机制知之甚少。LINE元素是 人类基因组中特别丰富的一类逆转录因子，对基因表达有很大影响。 通过在转录启动子附近插入来表达。此外，LINE引起的异常反转录转位 这些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的逆转录转座水平。第二组内含子被认为是 真核非LTR逆转录元件的祖先，例如在人类中发现的LINE元件。组II 由于其稳定性和高精度，逆向单元是用于结构确定的更易处理的模型系统。 体外反转录转座活性。为了深入了解反转录转座的结构，我们的目标是使用单- 粒子cryo-EM解决高分辨率结构的第二组内含子逆向元件。具体来说，我们将获得 在反转录转座成dsDNA的不同阶段，反转录元件的结构。我们将使用一个 生物化学、遗传学和结构学方法的组合：1）确定自由基团的结构 II内含子逆转录元件。2)确定整合到靶双链DNA中的机制。这是 预期与哺乳动物逆转录元件催化的逆转录转座有直接的平行关系。我们将 捕捉反转录转座的不同阶段，以创建整个过程的“分子电影”。这将 代表了第一个反转录的原子模型。

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.