Post-translational regulation of recombinase function by intein splicing

内含肽剪接对重组酶功能的翻译后调节

• 批准号: 9315001
• 负责人: Christopher William Lennon
• 金额: $ 6.1万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9315001
• 关键词: Archaea; Bacteria; Bacterial Genome; Binding; Biochemical; Biochemistry; Biotechnology; Characteristics; Chemical Engineering; DNA; DNA Binding; DNA Damage; Escherichia coli; Essential Genes; Family; Genes; Genetic; Glycine decarboxylase; Human; In Vitro; Invaded; Mobile Genetic Elements; Molecular; Molecular Models; Monitor; Mycobacterium tuberculosis; Nature; Organism; Post-Translational Regulation; Process; Property; Protein Splicing; Proteins; Pyrococcus horikoshii; RNA Splicing; Rad51 recombinase; Regulation; Role; Site; Structural Biologist; System; Temperature; Thermometers; Work; base; collaborative environment; fungus; in vivo; intein; interdisciplinary approach; member; molecular modeling; new therapeutic target; novel; pathogen; protein function; recombinase; response; structural biology; tool

Abstract Inteins, or protein introns, invade genes at the level of DNA and are expressed within host proteins. Protein splicing occurs when the intein auto-catalytically excises itself from the host protein. Inteins are abundant in nature, present in approximately one half of archaeal and a fourth of bacterial genomes, but have been vastly understudied to date. Recent work has suggested that inteins may be more than selfish invaders, and in some instances may have evolved to regulate host protein function prior to splicing, representing a completely novel form of post-translational regulation. We have discovered that RadA from Pyrococcus horikoshii, a member of the highly conserved RecA/Rad51 recombinase family, splices dramatically faster in the presence of its substrate, ssDNA. This result represents the first example of a biomolecule critical to host protein function influencing splicing, and detailed study of this system may prove key to understanding the possible regulatory role of inteins. We seek to explore this provocative result further in vitro and in vivo both in E coli and the native organism P. horikoshii, using a combination of biochemistry, genetics, molecular modeling and structural biology. We also seek to exploit a previously described temperature- dependent splicing characteristic of P. horikoshii RadA to develop a fluorescent molecular thermometer, which will both inform our understanding of the interactions between intein and RadA prior to splicing and serve as a tool for biotechnology. These interdisciplinary approaches will occur in a collaborative environment with structural biologists and chemical engineers.

抽象的 内膜或蛋白质内含子在DNA水平上侵入基因，并在宿主蛋白中表达。蛋白质 当内部自动催化物从宿主蛋白中自身切除时，就会发生剪接。 inteins很丰富 自然，大约有一半的古细菌和四分之一的细菌基因组，但已经存在 迄今为止已经大大研究了。最近的工作表明，Inteins可能不仅仅是自私入侵者，而且 在某些情况下，可能已经演变成在拼接之前调节宿主蛋白功能，代表 完全新颖的翻译后调节形式。我们已经发现了来自pyrococcus的RADA Horikoshii是高度保守的RECA/RAD51重组酶家族的成员 其底物ssDNA的存在。该结果代表了至关重要的生物分子的第一个例子 宿主蛋白质功能影响剪接以及对该系统的详细研究可能是理解的关键 输入的可能的调节作用。我们试图进一步探索这种挑衅性结果的体外和体内 在e大肠杆菌和原生生物p。horikoshii中，都结合了生物化学，遗传学， 分子建模和结构生物学。我们还试图利用先前描述的温度 P. horikoshii Rada的依赖性剪接特征，形成荧光分子温度计， 这两个都将使我们在拼接和RADA之间的相互作用之前了解我们的理解和 充当生物技术的工具。这些跨学科的方法将在协作中发生 结构生物学家和化学工程师的环境。