Sense/Antisense Genetic Coding and the Origins of Translation

描述（由申请人提供）：功能进化的研究具有挑战性，因为它需要为已灭绝的祖先节点重建合理的模型。我们建议建立实验可测试的模型，用于研究进化如何在蛋白质水平上引入和修改与适应性增加相关的功能关系。我们补充了从序列系统发育（祖先基因复活）中建立的统计推断，并采用了类似的，但更激进的程序，该程序基于识别共同的核心三级结构来重建远比基于系统发育序列的方法更古老（尽管不太安全）的酶的基因结构和功能。我们专注于非常古老的祖先氨基酰基trna合成酶模型，其进化血统是密码子导向蛋白质合成和基因表达起源的关键。这些aaRS并不都是同源的，而是出现在两个不同的超家族中。这个项目最深层的动机是想要了解这两个超家族之间的深刻对称性。在几个假设中，我们希望测试的是一类和二类AARS的祖先形式最初是在相同的义/反义开放阅读框架的相反链上编码的。我们引入Urzymology（源自Ur = primitive, original, early + enzyme）一词来描述这类祖先蛋白的创造和实验研究，这是祖先基因复活所无法达到的。urzyymology带来了跨越时间操纵生物物体的能力。Urzymes与随后获得的功能模块之间的互补以及Urzymes与当代酶的平行诱变使得测试催化、特异性和变构进化的明确模型成为可能。已发表的关于许多明显偶然性的原理证明提供了变革性研究的异常强大的组合。目的1将记录I类和II类氨基酰基trna合成酶Urzymes的相对氨基酸特异性，并建立Urzymes与当代aaRS之间的详细机制差异。目的2致力于实验研究罗丁-大野假说，即两个aaRS类出现在同一祖先基因的相反链上。目的3将加强计算设计过程和建立遗传系统，以选择和表征较少的细胞毒性结构，最终用于选择具有改进酶功能的Urzymes。通过这些实验绘制功能适应的记录将通过提供实验工具来获取和表征可能的进化中间产物，从而补充不断增长的基因组序列数据库。概述功能适应的进化记录将通过对蛋白质中模块如何相互作用的明确的新理解来补充序列数据库的直观使用，以及补充药物设计和新蛋白质试剂的工程和设计的实验范式。通过识别同时出现的蛋白质超家族对，验证正义/反义遗传编码将丰富对蛋白质组的理解，增强“同源性”的意义。

DESCRIPTION (provided by applicant): The evolution of function is challenging to study, because it requires reconstructing reasonable models for extinct ancestral nodes. We propose to generate experimentally testable models for studying how evolution has introduced and modified functional relationships at the protein level associated with increased fitness. We complement the established statistical inference from sequence phylogenies (ancestral gene resurrection) with an analogous, but more radical procedure based on identifying common, core tertiary structures to reconstruct gene structure and function of enzymes far more ancient (albeit less secure) than those accessible from phylogenetic sequence-based methods. We focus on very ancient models for ancestral aminoacyl-tRNA synthetases, whose evolutionary descent was key to the origins of codon-directed protein synthesis and hence gene expression. The aaRS are not all homologous, but instead occur in two distinct superfamilies. This project is most deeply motivated by a desire to understand the profound symmetries that relate the two superfamilies. Among several hypotheses we hope to test is that the ancestral forms of class I and class II AARS were initially encoded on opposite strands of the same sense/antisense open reading frame. We introduce the term Urzymology (from Ur = primitive, original, early + enzyme) to describe the creation and experimental study of such ancestral proteins, which lie beyond the reach of ancestral gene resurrection. Urzymology brings with it the ability to manipulate biological objects across time. Complementation between Urzymes and subsequently acquired functional modules and parallel mutagenesis of Urzymes and contemporary enzymes make it possible to test explicit models for the evolution of catalysis, specificity, and allostery. Published proofs-of-principle for many obvious contingencies provide an exceptionally strong combination of transformative research. Aim 1 will document the relative amino acid specificities of Class I and II aminoacyl-tRNA synthetase Urzymes, and establish detailed mechanistic differences between the Urzymes and contemporary aaRS. Aim 2 is devoted to experimental study of the Rodin-Ohno hypothesis that the two aaRS classes arose on opposite strands of the same ancestral gene. Aim 3 will enhance the computational design process and establish genetic systems to select and characterize less cytotoxic constructs for eventual use in selecting Urzymes with improved enzymatic function. Charting the record of functional adaptation with experiments like those proposed here will complement the growing genomic sequence database by providing experimental tools to access and characterize likely evolutionary intermediates. Outlining the evolutionary record of functional adaptation will supplement intuitive use of sequence databases with experimental paradigms that complement drug design and the engineering and design of new protein reagents by explicit new understanding of how modules interact in proteins. Validating sense/antisense genetic coding would enrich understanding of the proteome, by identifying pairs of protein superfamilies that arose simultaneously, enhancing the meaning of "homology". PUBLIC HEALTH RELEVANCE: To examine how catalysis and specificity evolve, we recreate extinct proteins predicted by evolutionary analysis to be critical for protein synthesis. Examining functional evolutionary branch points experimentally in this manner will generate and test entirely new insights. Central to the effort is the increasing evidence that genes for the two aminoacyl-tRNA synthetase Classes were originally encoded sense and antisense, on opposite strands of the same ancestral gene. The sense/antisense coding hypothesis simplifies what appear to be irreducible complexities associated with the origins of translation. Experimental validation would significantly change the way we understand the proteome and provide new explanations for the existence, complexity, and elegance of the specific genes and systems that drive both normal and pathological biological processes.