Structural understanding of Mu transposition

Mu转置的结构理解

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：这项工作将结合晶体学和生物化学研究，以了解经典 DNA 转座酶（噬菌体 Mu 的转座酶现已成为生物技术工具）的组织和调节。这是转座酶和逆转录病毒整合酶的大型且高度多样化的“DDE”家族中最彻底的特征，对 Mu 的研究将继续阐明转化为该家族其他成员的范式。初步结果包括最近确定的活性 Mu 转座酶 - DNA 复合物的结构，其中包含 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、最复杂的结构，也是唯一与目标 DNA 一起结晶的第二个 DDE 重组酶。该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型。目前的结构指导构建一种活性但更紧凑的嵌合转座酶，该转座酶具有不同的 DNA 结合域，这可能有助于结晶，将促进我们实验室和其他实验室的体外实验，并可能有助于将该系统设计为更有用的系统。生物技术工具。目标 2 是了解稳定性增强和 ClpX 识别背后的构象变化。我们的结构提出了一个模型，说明转座酶如何利用产物结合能来推动等能化学反应，以及极其稳定的最终复合物如何优先被 ATP 依赖性解折叠酶 ClpX 识别。该目标使用本体和单分子 FRET 和 LRET，以及一些晶体学和溶液测定。目的 3 是了解具有完整左右噬菌体末端的组装的结构基础。尽管活性复合物仅需要 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各包含 3 个转座酶结合位点的不同阵列。我们的结构为可检验的假设提供了基础，这些假设涉及为什么左端与右端不同以及它如何合并到最终的复合体中。这项工作将涉及精心挑选的专门针对不同结合位点的突变体的复杂形成和活性测定。详细了解该系统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看似额外的重组酶结合位点提供信息丰富的示例。