Sense/Antisense Genetic Coding and the Origins of Translation

 描述（由申请人提供）：密码子依赖性翻译将晚期化学进化与生物进化联系起来，这是系统发育推断和遗传学的基础。氨酰-tRNA合成（阿尔斯）实际上是翻译代码。因此，对祖先阿尔斯的实验研究对当代分子生物学、遗传学、生物物理学和生物化学具有深刻和广泛的价值。以前的工作创造并广泛验证了一种新的方法来访问与翻译起源相关的事件和过程。实验方法使用Urzymes，其是从蛋白质超家族的不变核心开发的构建体。代表I类和II类（阿尔斯）的祖先形式的Urzymes具有约15%的质量，但保留当代阿尔斯的60%的催化能力。该方法还引入了新的生物信息学，表明I类和II类阿尔斯来自同一基因的相反链。因此，多个有义/反义比对的密码子中间碱基配对（<MBP>）探针比使用祖先基因重建的那些可访问的系统发育关系更早。这项建议将利用这些新的工具来解决三个问题：（一）什么样的分子和过程引起了高度进化的复杂的I类和II Urzymes？<MBP>94个残基的I类和II类阿尔斯Urgenes的值将区分两条链特化的顺序，打破有义/反义编码的限制，并辐射以给出亚类，然后给出编码氨基酸的完整规范集。已通过诱变验证了来自设计的有义/反义基因的46-aa ATP结合位点的催化作用。我们将使用热力学循环来测量46聚体活性位点残基之间的偶联，并尝试使用还原的氨基酸字母表来构建它们。(II)Urzyme的结构和功能与当代酶有何不同？将在Urzymes和II类HisRS中测量tRNA特异性和长程能量偶联。NMR研究将确定Uryzmes是否是折叠的，熔融的球状或本质上无序的;稳定性和催化作用的温度依赖性将验证所得结论。(III)在执行遗传密码的过程中，Urzymes可能发挥了什么生物学作用？一种新的方法将使用阿尔斯敲除的构建来测试Urzymes是否可以补充阿尔斯敲除或者它们是否在体内有毒，并且将开发体外翻译系统，以最终概括从定义的组分开发规范遗传密码的步骤。以前的结果表明这些都是可行的。

 DESCRIPTION (provided by applicant): Codon-dependent translation connects late chemical evolution to biological evolution, which is the basis for phylogenetic inference and genetics. Aminoacyl-tRNA syntheses (aaRS) actually translate the code. Experimental study of ancestral aaRS is thus deeply and broadly valuable to contemporary molecular biology, genetics, biophysics, and biochemistry. Previous work created and extensively validated a new approach to accessing events and processes associated with the origins of translation. The experimental approach uses Urzymes, which are constructs developed from invariant cores of protein superfamilies'. Urzymes representing ancestral forms of Class I and II (aaRS) have ~15% of the mass but retain 60% of the catalytic proficiency of contemporary aaRS. The approach also introduced new bioinformatics to show that Class I and II aaRS descended from opposite strands of the same gene. Thus, codon middle-base pairing of multiple sense/antisense alignments (<MBP>) probes earlier phylogenetic relationships than those accessible using ancestral gene reconstruction. This proposal will exploit these new tools to address three questions: (I) What molecules and processes gave rise to the highly evolved sophistication of Class I and II Urzymes? <MBP> values for 94-residue Class I and II aaRS Urgenes will distinguish the order in which the two strands specialized, breaking the constraint of sense/antisense coding, and radiated to give subclasses and then a full canonical set of coded amino acids. Catalysis by 46-aa ATP binding sites from a designed sense/antisense gene have been validated by mutagenesis. We will use thermodynamic cycles to measure coupling between 46mer active-site residues and attempt to construct them using reduced amino acid alphabets. (II) How do Urzyme structures and functions differ from contemporary enzymes? tRNA specificities and long-range energetic coupling will be measured in Urzymes and in Class II HisRS. NMR studies will determine whether the Uryzmes are folded, molten globular or intrinsically disordered; the temperature dependence of stability and catalysis will verify the resulting conclusions. (III) What biological roles could the Urzymes have carried out in the course of implementing the genetic code? A novel method will use construction of aaRS knockouts to test whether Urzymes can complement aaRS knockouts or whether they are toxic in vivo and an in vitro translation system will be developed, to eventually recapitulate steps in the development of the canonical genetic code from defined components. Previous results imply these are all doable.