Coordination of translation and assembly of protein complexes

大部分活细胞的蛋白质组只有在参与寡聚蛋白复合物时才能获得功能，但对蛋白质复合物的组装过程知之甚少。在之前的资助期内，我们发现在所研究的六种细菌蛋白复合体中，有五种复合体的组装开始共翻译，一旦接触间期暴露在翻译核糖体表面，复合体的组装涉及亚基与新生的伙伴亚基链的结合。在利用luxA-luxB操纵子编码的异二聚体细菌荧光素酶进行的原理验证实验中，我们进一步发现luxA-luxB在多顺反子mRNA上的连续定位允许本地化翻译，促进大肠杆菌细胞中高效的复合体组装。这表明一种广泛的机制将蛋白质复合物的组装和功能直接与遗传编码和位置（物理）信息联系起来。这一概念上的进步现在提供了一个具有丰富监管潜力的领域，我们建议在即将到来的融资期内进行探索。我们将使用一组合适的蛋白质复合物，并将重点放在大肠杆菌中蛋白质复合物组装的机制方面，以及组装机制如何与主动翻译的核糖体整合。具体来说，我们将首先研究新生链相互作用本身。利用核糖体分析、生化和遗传方法，我们将研究与伙伴亚基的新生链相互作用的结构和动力学特征（与N. Budisa和M. Rodnina合作），并为我们的合作者R. Lipowsky（具体目标I至III）对组装反应的数学描述提供定量数据。然后，我们还将研究操纵子中的基因组织如何影响共翻译组装，并确定翻译速度对亚基组装的影响（Specific Aims IV和V）。这将阐明在翻译成三维蛋白质复合物组装过程中解码和传递的物理和遗传信息与细胞功能之间的相互作用。

A large percent of any proteome of living cells acquires function only when participating in oligomeric protein complexes, but little is known about the process of protein complex assembly. In the previous funding period, we established that in five of the six bacterial protein complexes examined, complex assembly begins co-translationally involving subunit association with nascent chains of partner subunits once the contact interphase is exposed on the surface of the translating ribosome. We further found in proof-of-principle experiments using heterodimeric bacterial luciferase encoded by the luxA-luxB operon, that contiguous positioning of luxA-luxB on the polycistronic mRNA allows localized translation promoting efficient complex assembly in E. coli cells. This suggests a widespread mechanism which links protein complex assembly and function directly to genetically encoded and positional (physical) information. This conceptual advance now provides an area rich with regulatory potential which we propose to explore in the upcoming funding period.We will use a set of suitable protein complexes, and focus on mechanistic aspects of protein complex assembly in E. coli and how the assembly mechanism integrates with the actively translating ribosome. Specifically, we will first investigate nascent chain interactions per se. Using ribosome profiling, biochemical and genetic methodologies, we will investigate structural and kinetic features of nascent chain interactions with partner subunits (in collaboration with N. Budisa and M. Rodnina), and provide quantitative data for a mathematical description of an assembly reaction by our collaborator R. Lipowsky (Specific Aims I to III). We will then also investigate how organization of genes in operons affects co-translational assembly and determine the effect of translation speed on subunit assembly (Specific Aims IV and V). This will elucidate the interplay between physical and genetic information decoded and transmitted during translation into 3-dimensional protein complex assembly, and cellular function.