Sense/Antisense Genetic Coding and the Origins of Translation

 描述(申请人提供)：依赖密码子的翻译将晚期的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。氨基酰-tRNA合成(AARs)实际上翻译了代码。因此，祖先AARs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远而广泛的价值。以前的工作创建并广泛验证了一种访问与翻译起源相关的事件和流程的新方法。实验方法使用Urzyme，这是从蛋白质超家族的不变核心发展而来的结构。代表第I类和第II类(AARs)祖先形式的Urzyme具有约15%的质量，但保留了当代AARs 60%的催化熟练程度。该方法还引入了新的生物信息学，以表明I类和II类AAR来自同一基因的相反链。因此，多个正义/反义比对的密码子中碱基配对(&lt；MBP&gt；)比使用祖先基因重建获得的系统发育关系更早。这项提议将利用这些新工具来解决三个问题：(I)是什么分子和过程导致了第I类和第II类Urzyme的高度进化的复杂性？&lt；MBP&gt；94个残基的第I类和第II类Aars Urgene的值将区分两条链专门化的顺序，打破正义/反义编码的限制，并辐射产生亚类，然后是一组完整的编码氨基酸。一个设计的正/反义基因的46-aaATP结合位点的催化作用已经通过突变得到验证。我们将使用热力学循环来测量46聚体活性中心残基之间的偶联，并试图使用还原的氨基酸字母表来构建它们。(Ii)Urzyme的结构和功能与同时代的酶有何不同？将在Urzyme和II类HisRs中测量tRNA的特异性和远程能量耦合。核磁共振研究将确定Uryzme是折叠的、熔融的球状的还是本质上无序的；稳定性和催化的温度依赖性将验证由此得出的结论。(3)在实施遗传密码的过程中，Urzyme可能发挥了哪些生物学作用？一种新的方法将使用构建Aars基因敲除来测试Urzyme是否可以补充Aars基因敲除，或者它们在体内是否有毒，并将开发一个体外翻译系统，最终概括从定义的成分发展典型遗传代码的步骤。此前的结果表明，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.