Sense/Antisense Genetic Coding and the Origins of Translation

描述(申请人提供)：我们的长期目标是检验来自第I类和第II类氨基酰-tRNA合成酶(AARs)的核心结构的催化活性，从实验上检验蛋白质合成开始使用由同一基因的相反链编码的两个低特异性氨基酸激活酶的假设，其当代后代是10个第I类和第10个第II类AARs。先前关于转移RNA结构域的工作表明，受体茎微螺旋可以以全长tRNA的三个数量级内的速率被特异性氨基酰化，从而确立了tRNA进化的模块化。我们根据每一类约1900个多个序列比对中的序列熵重新检查了I类AAR的三级结构。以这种方式获得的I类超家族的新马赛克结构揭示了其序列来自Rossmann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的核心片段，以及来自核心催化域的氨基酸专一性决定螺旋。这种核心结构是模块化的，并且在所有I类AAR的十个家族中都是紧密不可叠加的。我们已经证明了一个“最小催化模块”，它是与Brian Kuhlman合作使用蛋白质设计方法从TrpRS中衍生出来的，是相当活跃的。我们的第一个目标是利用稳态动力学、活性位点突变来更全面地表征I类AARS最小催化结构域的这一活性，并通过构建最小催化结构域与马赛克层次结构中其他模块组件的组合来评估随后积累的模块的功能贡献，特别是反密码子结合和CP1插入结构域。我们的第二个目标是实现一个类似的策略来检查来自相应的II类AAR的最小催化结构域的催化活性。我们的目标是证明可以从两个AARS类中获得类似长度的活性片段，作为对该假说的实验支持。我们的第三个目标是使Kuhlman教授使用的蛋白质设计软件同时设计一对I类和II类最小催化结构域，在保持催化活性的同时改善其正义/反义编码。这项研究计划承诺不仅扩大对蛋白质合成起源中的一个重要事件的理解，还包括对蛋白质结构的正义/反义编码的约束。

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.