Interactions Regulating Translation and Protein Biogenesis in Vivo

描述（由申请人提供）： 正常的细胞功能需要细胞蛋白质的有效折叠。在细胞中，蛋白质折叠与核糖体的蛋白质合成同时开始，遵循与体外全长蛋白质重折叠不同的途径。E.大肠杆菌的翻译速率为~ 20 aa/sec，但对于特定mRNA序列的翻译，该速率可以变化超过一个数量级。最近的研究结果表明，改变一些基因的一小部分的翻译速率可以显著影响编码蛋白的折叠效率（正确折叠与错误折叠和聚集或降解），或改变两种替代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生物合成的潜在影响，但核糖体与mRNA和/或控制局部翻译速率的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哪部分蛋白质具有共翻译折叠机制，该机制会受到翻译速率改变的显着影响。该提案的PI构建了一个网络，以跨越其在遗传学，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专业知识，创建了一个独特的团队，适合解决有关E.杆菌我们的网络将决定：（1）mRNA和/或新生链序列的哪些特定特征在体内形成绝对局部翻译速率，以及通过哪些机制？(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)翻译停顿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？总之，从这一建议的结果将代表关键步骤的翻译速度控制在E。大肠杆菌，以及改变翻译速率对蛋白质生物合成的影响，包括人类蛋白质的异源表达。

DESCRIPTION (provided by applicant): Proper cellular function requires efficient folding of cellular proteins. In the cell, protein folding starts co-translationally, concomitantly with protein synthesis by the ribosome, following pathways that can be distinct from the refolding of full-length proteins in vitro. The average, bulk 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.