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: 7917117
• 负责人: Charles W. Carter
• 金额: $ 20.51万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917117
• 关键词: Active Sites; Address; Amino Acids; Amino Acids Activation; Amino Acyl-tRNA Synthetases; Anticodon; Base Sequence; Binding; Binding Sites; Bioinformatics; Biological; Biological Assay; Birth; C-terminal; Catalysis; Catalytic Domain; Code; Collaborations; Communities; Complement; Complex; Data; Dinucleoside Phosphates; Entropy; Enzymes; Event; Evolution; Family; Figs - dietary; Genes; Genetic Code; Goals; In Vitro; Joints; Kinetics; Length; Libraries; Ligase; Light; MHC Class I Genes; Measures; Messenger RNA; Methods; Monitor; Mutate; Mutation; Natural Selections; Ohio; Pathway interactions; Pattern; Peptides; Probability; Property; Protein Biosynthesis; Proteins; Relative (related person); Research; Research Personnel; Sequence Alignment; Side; Software Design; Soil; Solubility; Specificity; Staging; Structure; Testing; Transfer RNA; Transfer RNA Aminoacylation; Translations; Tryptophan; Universities; Validation; Variant; Vermont; Vision; Work; combinatorial; design; experience; improved; mutant; professor; programs; prospective; protein folding; protein structure; single molecule; stem; success; urinary gonadotropin fragment; virtual

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- tRNA合成酶（阿尔斯）的核心结构的催化活性，以实验测试蛋白质合成开始于使用由相同基因的相反链编码的两种低特异性氨基酸活化酶的假设，并且其当代后代是10种I类和10种II类阿尔斯.以前的工作转移RNA结构域表明，受体茎微螺旋可以特异性氨酰化的速率在三个数量级内观察到的全长tRNA，从而建立了tRNA进化的模块化。我们已经重新检查了I类阿尔斯的三级结构，在多个序列比对中的序列熵，每个类约1900。以这种方式获得的I类超家族的新镶嵌结构揭示了核心片段，其序列来源于来自Rossmann二核苷酸结合折叠的N-和C-末端b-a-B交叉连接的不连续片段，以及来自核心催化结构域的氨基酸特异性决定螺旋。这种核心结构是模块化的，并且在I类阿尔斯的所有10个家族中是紧密重叠的。我们已经证明，一个“最小的催化模块”，来自TrpRS使用蛋白质设计方法与Brian Kuhlman合作，是相当活跃的。我们的第一个目标是更充分地表征这种活性的I类阿尔斯最小催化结构域，使用稳态动力学，活性位点突变，并通过构建最小催化结构域与其他模块组件的组合从镶嵌层次结构，特别是反密码子结合和CP 1插入结构域，以评估随后积累的模块的功能贡献。我们的第二个目标是实施一个类似的策略来检查催化活性来自相应的最小催化结构域从II类阿尔斯。我们的目标是证明，活性片段相似的长度可以来自两个阿尔斯类作为实验支持的假设。我们的第三个目标是调整Kuhlman教授使用的蛋白质设计软件，同时设计保留催化活性的I类和II类最小催化结构域对，同时改善其有义/反义编码。这项研究计划不仅有望扩展对蛋白质合成起源中的重要事件的理解，而且还将扩展对蛋白质结构的有义/反义编码中的约束的理解。