Structural understanding of Mu transposition

Mu转置的结构理解

• 批准号: 9003060
• 负责人: PHOEBE A RICE
• 金额: $ 29.6万
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003060
• 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; Staging; Structure; System; Testing; Translating; Transposase; VDJ Recombinases; Variant; Work; base; biochemical tools; 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（由申请人提供）：这项工作将结合联合收割机晶体学和生物化学研究，以了解组织和一个经典的DNA转座酶，现在是一个生物技术工具，噬菌体穆调控。这是转座酶和逆转录病毒整合酶的大型和高度多样性的“DDE”家族的最彻底的特征，Mu的研究将继续阐明转化到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，其含有240 kDa的蛋白质和135 bp的DNA。这是迄今为止任何转座酶-DNA结构中最大和最复杂的结构，并且只有第二个DDE重组酶与靶DNA一起结晶。这一结构是进一步研究的跳板。 目的1是完成目前的结构，并获得尽可能高的分辨率晶体形式。目前的结构指导了一种活性但更紧凑的嵌合转座酶的构建，该转座酶具有不同的DNA结合结构域，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工程化为更有用的生物技术工具。 目的2是了解增加稳定性和ClpX识别的基础构象变化。我们的结构表明了一个模型，转座酶如何使用产物结合能来推动一个等能的化学反应，以及如何通过ATP依赖性解折叠酶ClpX优先识别极其稳定的最终复合物。该目标使用本体和单分子FRET和LRET，以及一些晶体学和溶液分析。 目的3是了解左右两端完整的噬菌体组装的结构基础。虽然一个活性复合物只需要4个转座酶拷贝（这是我们已经结晶的），噬菌体的右端和左端含有不同的3个转座酶结合位点的阵列。我们的结构提供了一个可检验的假设的基础，关于为什么左端与右端不同，以及它是如何被纳入最终的复合体。这项工作将涉及复杂的形成和活性测定与精心挑选的突变体专门针对不同的结合位点。对该系统的详细理解将为理解为什么许多其他移动的DNA元件具有不同的左末端和右末端以及额外的重组酶结合位点提供信息性实例。