Genetic Approaches to Protein-Nucleic Acid Interactions

蛋白质-核酸相互作用的遗传学方法

• 批准号: 7729084
• 负责人: PAUL R SCHIMMEL
• 金额: $ 40.35万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-03-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7729084
• 关键词: Active Sites; Aging; Alanine; Alanine-tRNA Ligase; Algorithms; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Anticodon; Apoptosis; Ataxia; Bacteria; Biological; Cell Aging; Cell Death; Cells; Cerebellum; Codon Nucleotides; Confusion; DNA Repair; DNA biosynthesis; Defect; Development; Disease; Embryo; Enzymes; Evolution; Fibroblasts; Foundations; Future; Genetic; Genetic Code; Genome; Glycine; Goals; Grant; Human; In Vitro; Incidence; Investigation; Laboratories; Lead; Learning; Life; Ligase; Link; Malignant Neoplasms; Mammalian Cell; Molecular; Molecular Chaperones; Mus; Mutation; Nerve Degeneration; Nucleic Acids; Oncogenic; Organism; Pathology; Pathway interactions; Phenotype; Population; Property; Protein Biosynthesis; Proteins; Purkinje Cells; RNA; Role; Series; Serine; Site; System; Time; Transfer RNA; Transfer RNA Aminoacylation; Translations; Triplet Multiple Birth; Up-Regulation; Ursidae Family; Work; biological systems; catalyst; cell age; design; in vivo; interest; polypeptide; programs; public health relevance; repaired; research study; response; tumorigenesis

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.

描述（由申请人提供）：该项目的重点是翻译装置的组件——氨酰 tRNA 合成酶——如何与翻译之外的生物途径和疾病联系起来。合成酶催化转移RNA的氨酰化，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。氨酰化中的小错误会导致错误翻译，即将错误的氨基酸掺入不断增长的多肽链中。这些错误通常可以通过合成酶的编辑活动来纠正，从而将错误的氨基酸从其所附着的 tRNA 中清除。合成酶中的编辑位点在进化中高度保守，这种保守性说明了它们的重要性。由于含有错误的蛋白质会随着时间的推移而积累，破坏编辑的突变对细菌来说是有毒的。在最后的资助期内，哺乳动物细胞被证明比细菌对氨酰化错误更加敏感。具有编辑缺陷的细胞以反式显性方式被驱动进入细胞凋亡样反应。此外，特定 tRNA 合成酶活性的轻微突变被证明会导致小鼠神经变性，从而导致共济失调等表型。这种轻微的编辑缺陷导致未折叠蛋白反应强烈上调，并导致小脑浦肯野细胞变性。进一步的研究表明，由于 DNA 复制装置（包括 DNA 修复系统）中蛋白质的错误翻译，编辑缺陷会导致衰老细菌发生突变。在这些最新结果的基础上，未来的实验旨在研究编辑缺陷如何导致衰老细胞中癌症的发展（致癌转化）。其他工作旨在了解哺乳动物细胞克服困难以避免将丝氨酸和甘氨酸与丙氨酸混淆的关键机制。这种混淆在一定程度上是通过一个具有编辑能力的基因组编码片段来解决的，该片段与特定合成酶的编辑域同源，并且可以反式作用以清除错误地带有丝氨酸或甘氨酸而不是丙氨酸的 tRNA。尽管编辑熟练的基因组编码片段的活性已在体外得到证实，但体内活性的重要性仍有待确定，这是下一个资助期的目标。此外，该片段与分子共伴侣融合。融合的共伴侣部分如何调节编辑活动，以及融合如何将编辑活动连接到其他生物系统，是未来计划的重点。 公共健康相关性：该项目的重点是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该机制崩溃时可能导致的疾病类型。