Structural understanding of Mu transposition

Mu转置的结构理解

• 批准号: 8831212
• 负责人: PHOEBE A RICE
• 金额: $ 1.8万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8831212
• 关键词: Address; Antibiotic Resistance; Architecture; Bacteriophages; Binding; Binding Sites; Biochemical; Biological Assay; Biotechnology; C-terminal; Catalytic Domain; Chimera organism; Collaborations; Complex; Crystallization; Crystallography; DNA; DNA Binding; DNA Binding Domain; DNA Structure; DNA Transposons; DNA-Protein Interaction; Data Set; Dissection; Drops; Elements; Engineering; Enhancers; Enzymes; Evolution; Family; Family member; Fluorescence Resonance Energy Transfer; HIV; In Vitro; Integrase; Investigation; Left; Measures; Mobile Genetic Elements; Modeling; Nature; Pathway interactions; Peptide Hydrolases; Physiological; Play; Protein Engineering; Proteins; Protomer; Reaction; Regulation; Resolution; Rest; Retroviridae; Role; Solutions; Staging; Structure; System; Testing; Translating; Transposase; VDJ Recombinases; Variant; Work; base; chemical reaction; complex R; fluorophore; improved; in vitro Assay; interest; luminescence resonance energy transfer; mu transposase; mutant; prevent; protein protein interaction; public health relevance; rapid technique; recombinase; reconstruction; research study; screening; single molecule; single-molecule FRET; tool; transposon/insertion element; unfoldase; vector

DESCRIPTION (provided by applicant): This work will combine crystallographic and biochemical studies to understand the organization and regulation of a classic DNA transposase that is now a biotech tool, that of bacteriophage Mu. This is the most thoroughly characterized of the large and highly diverse "DDE" family of transposases and retroviral integrases, and studies of Mu will continue to illuminate paradigms that translate to the rest of the family. Preliminary results include a recently-determined structure of an active Mu transposase - DNA complex, which contains 240 kDa of protein and 135bp of DNA. This is the largest and most complex structure of any transposase-DNA structure to date, and only the 2nd DDE recombinase to be crystallized with target DNA. This structure is serving as the springboard for further studies. AIM 1 is to complete the current structure and to obtain the highest resolution crystal form possible. The current structure guided the construction of an active but more compact chimeric transposase with a different DNA binding domain that may aid crystallization, will facilitate in vitro experiments in our lab and others', and may aid in engineering the system to be more useful as a biotech tool. AIM 2 is to understand the conformational changes that underlie increasing stability and ClpX recognition. Our structure suggests a model for how product binding energy is used by transposases to drive forward an otherwise isoenergetic chemical reaction, and how the extremely stable final complex can be preferentially recognized by the ATP-dependent unfoldase ClpX. This aim uses bulk and single-molecule FRET and LRET, as well as some crystallography and solution assays. AIM 3 is to understand the structural basis for assembly of with full left and right phage ends. Although an active complex requires only 4 copies of the transposase (which is what we have crystallized), the right and left ends of the phage contain different arrays of 3 transposase binding sites each. Our structure provides a basis for testable hypotheses regarding why the left end differs from the right and how it is incorporated into the final complex. This work will involve complex formation and activity assays with carefully chosen mutants specifically targeted to different binding sites. A detailed understanding of this system will provide an informative example for understanding why many other mobile DNA elements have different left and right ends, with seemingly-extra recombinase binding sites.

描述(申请人提供)：这项工作将结合结晶学和生化研究，以了解一种经典的DNA转座酶的组织和调控，它现在是一种生物技术工具，即噬菌体Mu。这是转座酶和逆转录病毒整合酶组成的大型、高度多样化的“DDE”家族最彻底的特征，对Mu的研究将继续阐明转化为该家族其他成员的范例。初步结果包括最近确定的具有活性的Mu转座酶-DNA复合体的结构，该复合体包含240 kDa的蛋白质和135bp的DNA。这是迄今为止所有转座酶-DNA结构中最大和最复杂的结构，也是第二个与目标DNA结晶的DDE重组酶。这一结构是进一步研究的跳板。目标1是完成当前的结构，并获得尽可能高分辨率的晶体形式。目前的结构指导了具有不同DNA结合域的活性但更紧密的嵌合转座酶的构建，这可能有助于结晶，将有助于我们和其他实验室的体外实验，并可能有助于工程系统作为生物技术工具更有用。目标2是了解稳定性增加和ClpX识别的基础构象变化。我们的结构建议了一个模型，说明转座酶如何利用产物结合能来推动其他等能的化学反应，以及极其稳定的最终络合物如何能够被依赖于ATP的展开酶ClpX优先识别。这一目标使用了整体和单分子FRET和LRET，以及一些结晶学和溶液分析。目的3是了解具有完整左右两端的噬菌体组装的结构基础。虽然活性复合体只需要4个转座酶拷贝(这是我们已经结晶的)，但噬菌体的左右两端分别包含3个转座酶结合位点的不同阵列。我们的结构为可测试的假说提供了基础，这些假说关于为什么左端不同于右端以及它如何被结合到最终的复合体中。这项工作将涉及复杂的形成和活性分析，精心选择的突变体专门针对不同的结合位点。对这一系统的详细了解将为理解为什么许多其他移动DNA元件具有不同的左端和右端，以及似乎额外的重组酶结合位点提供了一个信息丰富的例子。