Genetic Approaches to Protein-Nucleic Acid Interactions

描述(由申请人提供)：这个项目的重点是翻译装置的组成部分--氨基酰tRNA合成酶--如何与翻译之外的生物途径和疾病相联系。合成酶催化转移RNA的氨基酰化，从而建立三核苷酸密码子和氨基酸之间的遗传密码关系。这些酶出现在进化的早期，对所有生命形式都是必不可少的。氨酰化过程中的小错误会导致误翻译，即错误的氨基酸被加入到一个不断增长的多肽链中。这些错误通常通过编辑合成酶的活动来纠正，从而将错误荷电的氨基酸从其连接的tRNA中清除。合成酶中的编辑位点在进化中高度保守，这种保守说明它们是必不可少的。由于含有错误的蛋白质随着时间的推移而积累，扰乱编辑的突变对细菌是有毒的。在最后一次授权期，哺乳动物细胞被证明比细菌对氨酰化错误更敏感。带有编辑缺陷的细胞以一种跨显性的方式被驱动进入类凋亡反应。此外，一种特定的tRNA合成酶活性的轻微突变被证明会导致小鼠的神经退化，在其他表型中，这会导致共济失调。这种轻微的编辑缺陷导致未折叠蛋白反应的强烈上调，并导致小脑浦肯野细胞的退化。进一步的研究表明，编辑缺陷在老化的细菌中是突变的，因为DNA复制装置中的蛋白质翻译错误，包括DNA修复系统。在这些最近结果的基础上，未来的实验旨在调查编辑缺陷如何导致老化细胞中的癌症(致癌转化)的发展。其他工作旨在了解哺乳动物细胞克服挣扎以避免将丝氨酸和甘氨酸混淆为丙氨酸的关键机制。这种混乱在一定程度上是通过编辑熟练的基因组编码片段来处理的，该片段与特定合成酶的编辑域同源，并且可以反式作用于清除错误地含有丝氨酸或甘氨酸而不是丙氨酸的tRNA。尽管精通编辑的基因组编码片段的活性已在体外得到证实，但体内活性的意义仍有待确定，这是下一批赠款的目标。此外，该片段还与分子辅助伴侣融合。Fusion的辅助伴侣部分如何调控编辑活动，以及Fusion如何将编辑活动与其他生物系统连接起来，是未来计划中非常感兴趣的问题。 与公共卫生相关：该项目关注蛋白质合成错误如何导致细胞死亡和疾病。重点是了解避免错误的关键机制，以及当该机制故障时可能导致的疾病种类。

DESCRIPTION (provided by applicant): This project is focused on how components of the translation apparatus--aminoacyl tRNA synthetases-are connected to biological pathways outside of translation and to disease. The synthetases catalyze aminoacylation of transfer RNAs and thereby establish the genetic code relationship between trinucleotide codons and amino acids. The enzymes appeared early in evolution and are essential to all life forms. Small errors in aminoacylation result in mistranslation, that is, the incorporation of the wrong amino acid into a growing polypeptide chain. These errors are normally corrected by editing activities of synthetases, whereby a mischarged amino acid is cleared from the tRNA to which it is attached. The editing sites in synthetases are highly conserved in evolution, and this conservation speaks to their being essential. Because of the build up over time of proteins containing errors, mutations that disrupt editing are toxic to bacteria. Mammalian cells were shown in the last grant period to be far more sensitive than bacteria to errors of aminoacylation. Cells harboring an editing defect are driven into an apoptosis-like response in a trans-dominant way. In addition, a mild mutation in the activity of a specific tRNA synthetase was shown to cause neurodegeneration in mouse that, among other phenotypes, resulted in ataxia. This mild editing defect generated strong upregulation of the unfolded protein response and caused degeneration of Purkinje cells of the cerebellum. Further work showed that editing defects are mutagenic in aging bacteria, because of mistranslation of proteins in the DNA replication apparatus, including the DNA repair systems. Moving forward on the foundation of these recent results, future experiments are designed to investigate how editing defects can lead to the development of cancer (oncogenic transformation) in aging cells. Other work is aimed at understanding a key mechanism by which mammalian cells overcome their struggle to avoid confusing serine and glycine for alanine. This confusion is dealt with, in part, by an editing-proficient genome-encoded fragment that is homologous to the editing domain of a specific synthetase, and that can act in trans to clear a tRNA that is mischarged with serine or glycine instead of with alanine. Although the activity of the editing-proficient genome-encoded fragment has been demonstrated in vitro, the significance of the activity in vivo remains to be established and is a goal of the next grant period. In addition, this fragment is fused to a molecular co-chaperone. How the co-chaperone moiety of the fusion regulates the editing activity, and how the fusion connects the editing activity to other biological systems, is of great interest in the future program. PUBLIC HEALTH RELEVANCE: This project is focused on how errors in protein synthesis lead to cell death and disease. The emphasis is on understanding a key mechanism for error-avoidance and the kinds of diseases that can result when this mechanism breaks down.