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: 7132215
• 负责人: Charles W. Carter
• 金额: $ 27.32万
• 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 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7132215
• 关键词: active sites; aminoacid tRNA ligase; biochemical evolution; chemical kinetics; chemical models; chemical stability; computer simulation; enzyme activity; functional /structural genomics; gene mutation; genetic translation; molecular dynamics; nucleic acid sequence; peptide chemical synthesis; protein biosynthesis; protein structure function; transfer RNA

DESCRIPTION (provided by applicant): Our long-range goal is to examine catalytic activities of core structures derived from class I and II aminoacyl- tRNA synthetases (aaRS), to test experimentally the hypothesis that protein synthesis began using two low- specificity amino acid activating enzymes coded by opposite strands of the same gene, and whose contemporary progeny are the ten class I and ten class II aaRS. Previous work on transfer RNA domains showed that acceptor stem minihelices can be specifically aminoacylated at rates within three orders of magnitude of those observed for the full-length tRNAs and thereby established the modularity of tRNA evolution. We have reexamined class I aaRS tertiary structures in the light of sequence entropies in multiple sequence alignments of approximately 1900 for each class. A new mosaic structure of the class I superfamily obtained in this manner reveals a core fragment whose sequences derive from discontinuous fragments of the N- and C- terminal b-a-b crossover connections from the Rossmann dinucleotide-binding fold, together with the amino acid specificity-determining helix from the core catalytic domain. This core structure is both modular and closely superimposible in all ten families of class I aaRS. We have demonstrated that a "minimal catalytic module", derived from TrpRS using protein design methods in collaboration with Brian Kuhlman, is quite active. Our first goal is to characterize this activity more fully for class I aaRS minimal catalytic domains, using steady state kinetics, active site mutation, and to evaluate the functional contributions of subsequently accumulated modules by constructing combinations of the minimal catalytic domains with other modular components from the mosaic hierarchy, notably the anticodon binding and CP1 insertion domains. Our second aim is to implement a similar strategy to examine catalytic activities derived from corresponding minimal catalytic domains from class II aaRS. Our goal is to demonstrate that active fragments of similar length can be derived from both aaRS classes as experimental support for the hypothesis. Our third aim is to adapt the protein design software used by Professor Kuhlman to simultaneously design pairs of class I and class II minimal catalytic domains that retain catalytic activity while improving their sense/antisense encoding. This research program promises to extend understanding not only of an important event in the origin of protein synthesis, but also constraints involved in sense/antisense coding of protein structures.

描述（由申请人提供）：我们的长期目标是检查源自 I 类和 II 类氨酰基-tRNA 合成酶 (aaRS) 的核心结构的催化活性，以通过实验检验蛋白质合成开始使用两种低特异性氨基酸的假设由同一基因的相反链编码的激活酶，其当代后代是十个 I 类和十个 II 类 aaRS。先前关于转移RNA结构域的研究表明，受体茎小螺旋可以以全长tRNA观察到的三个数量级内的速率被特异性氨酰化，从而建立了tRNA进化的模块性。我们根据每类大约 1900 的多个序列比对中的序列熵重新检查了 I 类 aaRS 三级结构。以这种方式获得的 I 类超家族的新嵌合结构揭示了一个核心片段，其序列源自罗斯曼二核苷酸结合折叠的 N 端和 C 端 b-a-b 交叉连接的不连续片段，以及氨基酸特异性决定来自核心催化结构域的螺旋。该核心结构既是模块化的，又可以在 I 类 aaRS 的所有 10 个系列中紧密叠加。我们已经证明，与 Brian Kuhlman 合作使用蛋白质设计方法从 TrpRS 衍生的“最小催化模块”非常活跃。我们的第一个目标是使用稳态动力学、活性位点突变更全面地表征 I 类 aaRS 最小催化结构域的这种活性，并通过构建最小催化结构域与其他模块化组件的组合来评估随后积累的模块的功能贡献马赛克层次结构，特别是反密码子结合域和 CP1 插入域。我们的第二个目标是实施类似的策略来检查来自 II 类 aaRS 的相应最小催化结构域的催化活性。我们的目标是证明相似长度的活性片段可以从两个 aaRS 类别中衍生出来，作为该假设的实验支持。我们的第三个目标是采用 Kuhlman 教授使用的蛋白质设计软件来同时设计 I 类和 II 类最小催化结构域对，这些结构域保留催化活性，同时改进其有义/反义编码。该研究计划不仅有望扩展对蛋白质合成起源中重要事件的理解，而且还能扩展对蛋白质结构有义/反义编码所涉及的限制的理解。