Interactions Regulating Translation and Protein Biogenesis in Vivo

描述(由申请人提供)： 细胞的正常功能需要细胞蛋白质的有效折叠。在细胞中，蛋白质折叠开始于共翻译，伴随着核糖体的蛋白质合成，遵循与体外全长蛋白质重折叠不同的途径。在大肠杆菌中，BUL的平均翻译速率为-20aa/秒，但对于特定mRNA序列的翻译，这一速率可能会变化超过一个数量级。最近的结果强调，改变一些基因小部分的翻译率可以显著影响编码蛋白质的折叠效率(正确的折叠与错误折叠和聚集，或降解)，或改变两个可选折叠结构之间的分配。然而，尽管局部翻译速率对体内蛋白质的生物发生有潜在的影响，但控制局部翻译速率的核糖体与mRNA和/或新生链序列之间的相互作用仍然不透明。我们也不知道蛋白质组中有多少蛋白质具有共翻译折叠机制，这种机制会受到翻译速率变化的显著影响。这项提案的PI构建了一个网络，覆盖了他们在遗传学、分子生物学、基因组学、生物化学、生物物理学以及有机、分析和物理化学方面的现有专业知识，创建了一个独特的团队，专门解决关于大肠杆菌中翻译率改变的机制和结果的三个重大悬而未决的问题。我们的网络将决定：(1)mRNA和/或新生链序列的哪些特定特征决定了体内的绝对局部翻译速率，以及通过什么机制？(2)当翻译速率改变时，蛋白质组中的哪些蛋白质最容易聚集或降解？(3)翻译暂停在多大程度上改变了核糖体的构象和/或其与体内其他蛋白质的相互作用？综上所述，这一建议的结果将代表着朝着全面了解大肠杆菌中的翻译速率控制以及翻译速率改变对蛋白质生物发生的影响，包括人类蛋白质的异源表达的关键步骤。 公共卫生关注度： 蛋白质合成的速度并不一致，但我们并不完全理解为什么一些序列的翻译速度比其他序列慢。改变局部翻译速率会影响蛋白质折叠，从而影响细胞功能。我们将开发新的方法来测量局部翻译速率和决定这些速率的机制，并确定其折叠受翻译速率差异影响最大的蛋白质。

DESCRIPTION (provided by applicant): Proper cellular function requires efficient folding of cellular proteins. In the cell, protein foldng starts cotranslationally, concomitantly with protein synthesis by the ribosome, following pathways that can be distinct from the refolding of full-length proteins in vitro. The average, bul translation rate in E. coli is -20 aa/sec, but this rate can vary by more than an order of magnitude for the translation of specific mRNA sequences. Recent results have highlighted that altering the translation rate of small portions of some genes can significantly affect the folding efficiency of the encoded protein (correct folding versus misfolding and aggregation, or degradation), or alter the partitioning between two alternative folded structures. Yet despite the potential impact of local translation rate on protein biogenesis in vivo, the interactions between the ribosome and mRNA and/or nascent chain sequences that control local translation rate remain opaque. Nor do we know what fraction of proteins in the proteome has co-translational folding mechanisms that are significantly affected by altered translation rate. The Pis of this proposal have constructed a network to span their established expertise in genetics, molecular biology, genomics, biochemistry, biophysics, and organic, analytical and physical chemistry, creating a team uniquely suited to tackle three significant outstanding questions regarding the mechanisms and outcomes of altered translation rate in E. coli. Our network will determine: (1) What specific features of mRNA and/or nascent chain sequences shape absolute local translation rate in vivo, and by what mechanisms? (2) What proteins in the proteome are most susceptible to aggregation or degradation when translation rate is altered? (3) To what extent does translational pausing alter the conformation of the ribosome and/or its interactions with other proteins in vivo? Taken together, results from this proposal will represent crucial steps towards a comprehensive picture of translation rate control in E. coli, and the effects of altered translation rate on protein biogenesis, including the heterologous expression of human proteins. PUBLIC HEALTH RELEVENCE: The rate of protein synthesis is not uniform, but we do not fully understand why some sequences are translated more slowly than others. Altering local translation rate can affect protein folding, and hence cell function. We will develop new approaches to measure local translation rates and the mechanisms that determine these rates, and identify proteins whose folding is most affected by translation rate differences.