Sense/Antisense Genetic Coding and the Origins of Translation

正义/反义遗传编码和翻译的起源

• 批准号: 8964980
• 负责人: Charles W. Carter
• 金额: $ 35.57万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964980
• 关键词: Active Sites; Acylation; Address; Amino Acids; Amino Acids Activation; Amino Acyl Transfer RNA; Amino Acyl-tRNA Synthetases; Anticodon; Base Pairing; Behavior; Binding; Binding Sites; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Assay; Biological Process; Biology; Biophysics; C-Peptide; Catalysis; Chemical Evolution; Chemicals; Code; Codon Nucleotides; Complement; Core Protein; Coupling; Data; Dependence; Development; Disease; Elements; Enzymes; Escherichia coli; Event; Evolution; Genes; Genetic; Genetic Code; Goals; In Vitro; Kinetics; Knock-out; Label; Length; Link; Measurable; Measures; Mediating; Metals; Methods; Modeling; Molecular; Molecular Biology; Mutagenesis; Mutation; Peptides; Phylogenetic Analysis; Process; Proteins; RNA, Transfer, Amino Acid-Specific; Reaction; Resources; Role; Seeds; Site; Solid; Specificity; Staging; Structure; System; Temperature; Testing; Thermodynamics; Transfer RNA; Transfer RNA Aminoacylation; Translating; Translations; Variant; Viral Vector; Work; base; catalyst; combinatorial; design; digital; gene synthesis; gene therapy; improved; in vivo; melting; novel; novel strategies; protein folding; public health relevance; reconstruction; research study; stem; tool

 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.

 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，因此对祖先aaRS的实验研究实际上是深入且深入的。先前的工作创建并验证了一种获取与起源相关的事件和过程的新方法，对当代分子生物学、遗传学、生物物理学和生物化学具有广泛的价值。实验方法使用 Urzymes，它是从代表 I 类和 II 类 (aaRS) 祖先形式的 Urzymes 的不变核心开发而来的，具有约 15% 的质量，但保留了当代的 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 aaRS 源自同一基因的相反链，因此密码子为中间碱基。多重正义/反义比对（<MBP>）的配对比使用祖先基因重建可探测到的系统发育关系更早。该提案将利用这些新工具来解决三个问题：（I）什么分子和过程导致了高度进化的复杂性。 I 类和 II 类 Urzymes 的 <MBP> 值对于 94 个残基 I 类和 II 类 aaRS Urgenes 将区分两条链特化的顺序，打破了有义/反义编码，并辐射产生亚类，然后通过诱变验证了设计的有义/反义基因的 46-aa ATP 结合位点的完整规范集。 46mer 活性位点残基之间的偶联并尝试使用简化的氨基酸字母表构建它们 (II) Urzyme 的结构和功能与当代酶的特异性和功能有何不同？ Urzyme 中的长程能量耦合将在 II 类 HisRS 中进行测量，这将确定 Uryzmes 是折叠的、熔融球状的还是本质上无序的；稳定性和催化作用的温度依赖性将验证所得到的结论。 Urzymes 在执行遗传密码的过程中可以发挥什么作用？一种新方法将使用 aaRS 敲除的构建来测试 Urzymes 是否可以补充 aaRS 敲除，或者它们是否具有体内毒性和毒性。将开发一个体外翻译系统，以最终概括从定义的组件开发规范遗传密码的步骤。先前的结果表明这些都是可行的。