MOLECULAR BASIS FOR TRANSLATIONAL REGULATION

翻译调控的分子基础

• 批准号: 6174062
• 负责人: EDWARD CHU
• 金额: $ 20.27万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-13 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6174062
• 关键词: DNA footprinting; RNA binding protein; antineoplastics; crosslink; dihydrofolate reductase; enzyme activity; enzyme inhibitors; gel mobility shift assay; gene expression; genetic regulation; genetic regulatory element; genetic translation; messenger RNA; methotrexate; molecular genetics; mutant; protein biosynthesis; protein protein interaction; transcription factor; ultraviolet radiation

The long-term goal of this project is to characterize the regulation of gene expression at the translational level. As our model system, we will investigate the regulation of expression of dihydrofolate reductase (DHFR), a critical target in cancer chemotherapy. This enzyme catalyzes the NADPH-dependent reductive reaction which gives rise to the reduced folate, tetrahydrofolate, a key intermediate in one-carbon transfer reactions. As a result, DHFR is required for the de novo synthesis of purines and pyrimidines as well as for the synthesis of certain amino acids. Thus, DHFR plays a central role in maintaining the metabolic requirements of the cell. Previous studies from this lab have shown that in addition to its role in catalysis and cellular metabolism, DHFR also functions as an RNA binding protein. This protein binds with high affinity (3-5 nM) to its own DHFR mRNA, an interaction that results in the translational repression of DHFR mRNA with subsequent inhibition of synthesis of new DHFR protein. These studies demonstrate that the expression of DHFR is controlled at least, in part, by a translational autoregulatory feedback mechanism. This model of DHFR translational autoregulation would appear to have biological relevance in that it offers a rational mechanism for the tight control of DHFR expression within a given cell. However, treatment of DHFR protein with inhibitor compounds such as the antifolate analog methotrexate (MTX) alters the normal DHFR protein-DHFR mRNA interaction resulting in an enhanced translational efficiency of DHFR mRNA with an increased synthesis of new DHFR protein. Disruption of this normal regulatory process may provide an efficient mechanism for malignant cells to protect themselves in response to exposure to cytotoxic stress. To further our understanding of the molecular elements underlying the translational regulation of DHFR, two specific aims are proposed in this project: (1) Characterize the critical cis-acting elements on the DHFR mRNA that are required for this RNA-protein interaction. In this aim, we plan to identify the essential nucleotide sequences and/or secondary structural elements required for protein recognition, and (2) Characterize the critical trans-acting elements on the DHFR protein that are necessary for RNA binding. As part of this aim, we plan to identify the domain or domains on the DHFR protein as well as the critical amino acid contact points that mediate the process of RNA binding.

该项目的长期目标是表征翻译水平上基因表达的调控。 作为我们的模型系统，我们将研究二氢叶酸还原酶（DHFR）表达的调节，这是癌症化疗的关键靶点。 这种酶催化 NADPH 依赖性还原反应，产生还原叶酸、四氢叶酸，这是一碳转移反应的关键中间体。 因此，嘌呤和嘧啶的从头合成以及某些氨基酸的合成需要 DHFR。 因此，DHFR 在维持细胞代谢需求方面发挥着核心作用。 该实验室之前的研究表明，除了在催化和细胞代谢中的作用外，DHFR 还具有 RNA 结合蛋白的功能。 该蛋白以高亲和力 (3-5 nM) 与其自身的 DHFR mRNA 结合，这种相互作用导致 DHFR mRNA 的翻译抑制，随后抑制新 DHFR 蛋白的合成。 这些研究表明 DHFR 的表达至少部分受到翻译自动调节反馈机制的控制。 这种 DHFR 翻译自动调节模型似乎具有生物学相关性，因为它为严格控制给定细胞内的 DHFR 表达提供了合理的机制。 然而，用抗叶酸类似物甲氨蝶呤 (MTX) 等抑制剂化合物处理 DHFR 蛋白会改变正常的 DHFR 蛋白-DHFR mRNA 相互作用，导致 DHFR mRNA 的翻译效率增强，新 DHFR 蛋白的合成增加。 这种正常调节过程的破坏可能为恶性细胞提供一种有效的机制来保护自身以应对细胞毒性应激。为了进一步了解 DHFR 翻译调控的分子元件，本项目提出了两个具体目标：（1）表征 DHFR mRNA 上这种 RNA-蛋白质相互作用所需的关键顺式作用元件。 为此，我们计划鉴定蛋白质识别所需的必需核苷酸序列和/或二级结构元件，以及 (2) 表征 DHFR 蛋白上 RNA 结合所需的关键反式作用元件。 作为这一目标的一部分，我们计划鉴定 DHFR 蛋白上的一个或多个结构域以及介导 RNA 结合过程的关键氨基酸接触点。