Post-translational regulation of recombinase function by intein splicing

摘要 内含子，或蛋白质内含子，在DNA水平上入侵基因，并在宿主蛋白质中表达。蛋白 当内含素自动催化将自身从宿主蛋白质中切除时，剪接就会发生。Intein在 自然，存在于大约一半的古生菌和四分之一的细菌基因组中，但一直是 到目前为止，这方面的研究还远远不够。最近的研究表明，内含子可能不仅仅是自私的入侵者，而且 在某些情况下，可能已经进化到在剪接之前调节宿主蛋白质功能，代表着 全新的翻译后监管形式。我们发现从热球菌中分离出的RADA Horikoshii是高度保守的RecA/RAD51重组酶家族的成员，在 它的底物单链DNA的存在。这一结果代表了第一个生物分子对 宿主蛋白影响剪接的功能，对该系统的详细研究可能是理解这一系统的关键 内含子可能的调节作用。我们试图在体外和体内进一步探索这一挑衅的结果。 在大肠杆菌和原生生物P.horikoshii中，利用生物化学、遗传学、 分子建模和结构生物学。我们还试图利用之前描述的温度- Horikoshii Rada的依赖剪接特性研制荧光分子温度计， 这既将有助于我们理解内含素和RADA之间的相互作用，也有助于我们在剪接和 作为生物技术的一种工具。这些跨学科的方法将以协作的方式进行 环境与结构生物学家和化学工程师。

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.