Kinetic Discrimination of Substrates by the Ribosomal Biosynthetic Machinery

描述（由申请人提供）：翻译机制能够催化mRNA-模板指导的多肽合成，错误率仅为每1，000至10，000个合成的肽键中的一个。在过去的十年中，不仅在确定细菌翻译机器的高分辨率结构方面取得了重大进展，而且在翻译延长周期的精确机制方面也取得了重大进展，这使得第一次有可能研究翻译机器如何能够与化学上不同的20种天然氨基酸一起工作，但同时具有如此低的错误率。理解翻译机制的底物选择性是非常重要的，因为翻译对细胞功能至关重要，因为它涉及复杂的生物分子机器如何执行复杂功能的问题。尽管伸长周期的动力学模型提供了大量的数据，但几条证据表明，它不能完全解释底物的选择。虽然很明显，翻译机制不仅仅对密码子-反密码子相互作用敏感，但还没有一组决定性的实验从选择的角度来证明这种敏感性，也没有一个模型可以让我们预测这些其他因素如何影响翻译。我们目前处于填补这一空白的独特地位。目标1：我们建议测试的假设，氨酰-tRNA组合的歧视，在延长周期中使用肽掺入产率和竞争分析，我们已经开发了结合单分子FRET实验。目的2：我们建议测试的假设，歧视的氨酰-tRNA组合发生后，肽键的形成。我们还建议测试氨酰tRNA组合的哪些方面对于区分是重要的。文献中有一些早期发现表明，翻译水平的功能障碍与疾病病理学有关;例如，甘氨酸合成酶的突变与腓骨肌萎缩症有关。进一步阐明翻译机制的选择能力，将有助于我们理解翻译机制是如何处理这些故障的，或许还将揭示与病理学的其他重要联系。

DESCRIPTION (provided by applicant): The translation machinery is able to catalyze the mRNA-template directed synthesis of polypeptides with an error rate of only one in every 1,000 to 10,000 peptide bonds synthesized. Significant advances in the last decade not only in the determination of high resolution structures of the bacterial translation machinery but also the precise mechanism of the elongation cycle of translation make it possible for the first time to examine how the translation machinery is able to work with the chemically diverse 20 natural amino acids, but at the same time have such a low error rate. Understanding the substrate selectivity of the translation machinery is of major importance both because translation is essential to cellular function and because it speaks to the question of how complex biomolecular machines carry out sophisticated functions. Despite the amount of data provided by the kinetic model of the elongation cycle, several lines of evidence suggest that it cannot fully account for substrate selection. While it is apparent that the translation machinery is sensitive to more than just the codon anticodon interaction, there has not been a decisive set of experiments to demonstrate this sensitivity from the perspective of selection, nor is there a model that would allow us to predict how these various other factors would affect translation. We currently find ourselves in a unique position to fill this void. AIM 1: We propose to test the hypothesis that the aminoacy-tRNA combinations are discriminated during the elongation cycle using peptide incorporation yield and competition assays we've developed in conjunction with single molecule FRET experiments. AIM 2: We propose to test the hypothesis that discrimination of aminoacyl-tRNA combinations occurs after peptide bond formation. We also propose to test which aspects of the aminoacyl tRNA combinations are important for discrimination. There are some early findings in the literature indicating that malfunctions at the level of translation are involved disease pathology; for example, mutations in the glycine synthetase are linked to Charcot-Marie- Tooth disease. Further elucidation of the selection ability of the translation machinery will help us understand how these malfunctions are handled by the translation machinery, and will perhaps reveal other important links to pathology.