Sense/Antisense Genetic Coding and the Origins of Translation

描述（由申请人提供）：功能的进化研究具有挑战性，因为它需要为灭绝的祖先节点重建合理的模型。我们建议生成实验可测试的模型，用于研究进化如何在蛋白质水平上引入和修改与健身增加相关的功能关系。我们补充了已建立的统计推断序列同源性（祖先基因复活）与一个类似的，但更激进的程序的基础上确定共同的，核心的三级结构，以重建基因结构和功能的酶更古老（虽然不太安全）比那些访问系统发育序列为基础的方法。我们专注于非常古老的祖先氨酰-tRNA合成酶，其进化的后裔是关键的密码子指导的蛋白质合成的起源，因此基因表达的模型。阿尔斯并不都是同源的，而是出现在两个不同的超家族中。这个项目的最深动机是希望了解两个超家族之间深刻的对称性。我们希望检验的几个假设之一是，I类和II类阿尔斯的祖先形式最初是在相同的有义/反义开放阅读框架的相反链上编码的。我们引入术语Urzymology（来自Ur =原始的，原始的，早期的+酶）来描述这种祖先蛋白质的创造和实验研究，这些蛋白质超出了祖先基因复活的范围。Urzymology带来了跨时间操纵生物对象的能力。互补Urzymes和随后获得的功能模块和平行诱变Urzymes和当代酶之间的互补，使得有可能测试明确的模型的催化，特异性和变构的进化。针对许多显而易见的突发事件而发表的原理证明，提供了一个异常强大的变革性研究组合。目的1将记录I类和II类氨酰-tRNA合成酶Urzymes的相对氨基酸特异性，并建立Urzymes和当代阿尔斯之间的详细机制差异。目的2致力于Rodin-Ohno假说的实验研究，即两个阿尔斯类出现在相同祖先基因的相反链上。目标3将增强计算设计过程并建立遗传系统以选择和表征细胞毒性较小的构建体，以最终用于选择具有改善的酶功能的Urzyme。绘制功能适应的记录与实验，如这里提出的将补充不断增长的基因组序列数据库提供实验工具，以访问和表征可能的进化中间体。概述功能适应的进化记录将补充直观使用序列数据库的实验范式，补充药物设计和工程和设计新的蛋白质试剂的明确新的理解模块如何在蛋白质中相互作用。验证有义/反义遗传编码将丰富对蛋白质组的理解，通过识别同时出现的蛋白质超家族对，增强“同源性”的含义。 公共卫生相关性：为了研究催化和特异性是如何进化的，我们重新创建了进化分析预测的对蛋白质合成至关重要的灭绝蛋白质。以这种方式实验性地检查功能进化分支点将产生和测试全新的见解。这项工作的核心是越来越多的证据表明，这两类氨酰-tRNA合成酶的基因最初是在同一祖先基因的相反链上编码正义和反义的。有义/反义编码假说简化了与翻译起源相关的看似不可简化的复杂性。实验验证将显著改变我们对蛋白质组的理解，并为驱动正常和病理生物过程的特定基因和系统的存在、复杂性和优雅性提供新的解释。

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.