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: 7665311
• 负责人: Charles W. Carter
• 金额: $ 26.87万
• 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 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7665311
• 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）：我们的远程目标是检查源自I和II类氨基氨基氨基TRNA合成酶（AARS）的核心结构的催化活性，以实验测试蛋白质合成的假设，即蛋白质合成开始使用两个低特异性氨基酸激活酶的酶，该酶由同一基因II的相反群和其当代种类的相反链条编码，并且是当代的阶段，并且是当代的选择。先前关于转移RNA结构域的工作表明，可以在三个数量级以内对全长TRNA观察到的3个数量级的氨基酰基特异性氨基化液特异性，从而确定了TRNA演化的模块化。我们已经根据序列熵的序列以大约1900的序列熵的方式重新检查了I类三级结构。以这种方式获得的I级超家族的新的镶嵌结构揭示了一个核心碎片，其序列源自N-和c-末端B-A-B跨界的不连续片段，来自Rossmann Dinucleotide结合折叠的N-和C-末端B-A-B交叉连接，以及来自核心酸性螺旋的核心特异性螺旋，来自核心催化型催化性的螺旋。在I级AARS的所有十个家庭中，这种核心结构既模块化又非常叠加。我们已经证明，使用蛋白质设计方法与Brian Kuhlman合作衍生自TRPR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，主动位点突变，更全面地表征该活动的最小催化域，并通过构建与其他模块化成分的最小催化域的组合来评估随后累积模块的功能贡献，这些组合是从镶嵌层的其他模块化成分的组合，这一点既明显地构造cododon and cop1 intifion and cododiention and cpp1 compien and cop1 intering and cpp1 cpp1 compion intering and cop1。我们的第二个目的是实施类似的策略，以检查来自II类AAR的相应最小催化域而得出的催化活性。我们的目标是证明可以从两个AARS类中得出相似长度的主动片段作为对假设的实验支持。我们的第三个目的是调整Kuhlman教授使用的蛋白质设计软件，同时设计I类和II类最小催化域的一对，这些催化域保留了催化活性，同时改善其感官/反义编码。该研究计划有望扩大对蛋白质合成起源的重要事件的理解，而且还可以扩展蛋白质结构的感官/反义编码所涉及的限制。