Sense/Antisense Genetic Coding and the Origins of Translation

描述（由申请人提供）：我们的长期目标是研究从I类和II类氨基酰基tRNA合成酶（aaRS）衍生的核心结构的催化活性，通过实验验证蛋白质合成开始使用由同一基因的相反链编码的两种低特异性氨基酸激活酶的假设，其当代后代是10个I类和10个II类aaRS。先前对转移RNA结构域的研究表明，受体茎微螺旋的氨基酰化速率比全长tRNA的氨基酰化速率高3个数量级，从而建立了tRNA进化的模块化。我们根据每一类约1900个序列比对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类超家族的一个新的镶嵌结构揭示了一个核心片段，其序列来自Rossmann二核苷酸结合折叠中的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。这个核心结构是模块化的，并且在所有10个I类aaRS家族中都是紧密重叠的。我们已经证明，与Brian Kuhlman合作，使用蛋白质设计方法从trpr中获得的“最小催化模块”非常活跃。我们的第一个目标是利用稳态动力学、活性位点突变，更全面地表征I类aaRS最小催化结构域的活性，并通过构建最小催化结构域与马赛克结构中的其他模块组件的组合，特别是反密码子结合和CP1插入结构域，来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类似的策略来检查II类aaRS的相应最小催化域的催化活性。我们的目标是证明可以从两个aaRS类别中获得相似长度的活性片段，作为该假设的实验支持。我们的第三个目标是改编Kuhlman教授使用的蛋白质设计软件，同时设计I类和II类最小催化结构域对，这些结构域在保留催化活性的同时改善其正义/反义编码。这一研究项目不仅有望扩展对蛋白质合成起源的重要事件的理解，而且还将扩展对蛋白质结构的正义/反义编码的限制。

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.