Optimisation of heteromeric protein complex formation via co-ordinated mRNA localisation

该项目的目标是了解如何利用胞质翻译工厂和测试用例eIF3在酵母中最佳地生成异聚蛋白复合物。所获得的信息将用于生产在酵母中具有商业重要性的异源异聚复合物（例如血红蛋白和人促性腺激素）。但更广泛地说，所吸取的经验教训将普遍适用于使用酵母作为宿主生物体生产异源蛋白质。该学生奖学金的生物技术重点使其非常适合BBSRC高级别主题2“应对战略挑战”更具体地说，在该项目中，学生将研究mRNA到翻译工厂的协调定位和翻译。待研究的mRNA编码异聚蛋白复合物的亚基，并且待解决的关键问题是所得到的蛋白复合物的折叠、组装和生物学功能是否作为胞质翻译工厂中的确定位点处的生产的结果而受到调节。酵母中的eIF3、eIF2和NatA复合物将用作示例性蛋白质复合物，其中编码各种亚基的mRNA定位于翻译工厂。eIF3和eIF2代表参与蛋白质合成的关键复合物，其将核糖体募集到待翻译的mRNA中。NatA是一种N-乙酰基转移酶复合物，负责在核糖体产生蛋白质时对其进行修饰。这三种复合物都非常丰富和重要，因此它们在特定的地点共同形成并不奇怪，但不太清楚的是这种安排是如何建立，组织和控制的。这些研究将建立一套规则，然后可以应用于生产生物技术相关的异源蛋白质复合物-评估是否将异源mRNA靶向蛋白质工厂提高生产率和蛋白质复合物的产量。作为该项目的一部分，学生将接受大量不同方法和测定的培训。活细胞MS2为基础的成像技术和单分子荧光原位杂交将用于建立mRNA在细胞中的位置。创新的荧光分析将用于研究每个mRNA的翻译位点，多核糖体分析将用于验证翻译效率。生化分析将用于研究可溶性或蛋白质聚集体组分中存在的每个亚基的比例，并且使用酵母翻译提取物的体外分析将用于剖析eIF3组装过程。哪些RNA元件和蛋白质因子控制eIF3、eIF2和NatA mRNA的定位和翻译？2. eIF3、eIF2和NatA的产生是否受到改变mRNA定位的条件和突变的影响？3.翻译工厂的定位是否改变了蛋白质复合物亚基的比例，这些亚基是以可溶性活性形式存在的，而不是不溶性聚集体，尤其是在应激条件下？4.收集的eIF3，eIF2和NatA复合物形成的信息是否可以用于指导异源蛋白表达的翻译工厂的形成？

The goal of the project is to understand how to optimally generate heteromeric protein complexes in yeast making use of cytosolic translation factories and the test case eIF3. Information gained will be applied to the production of heterologous heteromeric complexes that can be commercially important in yeast (e.g. haemoglobin and human gonadotropin). More widely though the lessons learned will be generally applicable to the production of heterologous proteins using yeast as a host organism. The biotechnology focus of this studentship makes it a good fit to the BBSRC high-level theme 2 "Tackling strategic challenges"More specificially, in this project a student will study the co-ordinated localisation and translation of mRNAs to translation factories. The mRNAs to be studied encode subunits of heteromeric protein complexes, and the key question to be addressed is whether the folding, assembly and biological function of the resulting protein complexes are regulated as a consequence of production at a defined site in a cytosolic translation factory. The eIF3, eIF2 and NatA complexes in yeast will be used as exemplar protein complexes where the mRNAs encoding the various subunits are localised to translation factories. eIF3 and eIF2 represent key complexes involved in the synthesis of proteins in the recruitment of the ribosome to the mRNA to be translated. NatA is an N-acetyl transferase complex responsible for the modification of proteins while they are being produced by the ribosome. All three complexes are highly abundant and important, so it is unsurprising that they form co-translationally at specific sites, but what is less clear is how such an arrangement is set up, organised and controlled. These studies will establish a set of rules that can be then applied to the production of biotechnologically relevant heterologous protein complexes - assessing whether targeting heterologous mRNAs to protein factories increases productivity and yield of protein complexes.A student will be trained in a plethora of different methods and assays as part of this project. Live cell MS2-based imaging techniques and single molecule fluorescent in situ hybridisation will be used to establish the location of mRNAs in cells. Innovative fluorescent assays will be used to study the site of translation of each mRNA, and polysome analysis will be used to characterise the efficiency of translation. Biochemical assays will be used to study the proportion of each subunit that is present in the soluble or protein aggregate fractions, and in vitro assays using yeast translation extracts will be used to dissect the eIF3 assembly process.The questions that will be addressed are: 1. What RNA elements and protein factors control the localisation and translation of the eIF3, eIF2 and NatA mRNAs?2. Is eIF3, eIF2 and NatA production affected by conditions and mutations that alter the localisation of the mRNAs?3. Does localisation to a translation factory alter the proportion of protein complex subunits that are present as soluble active forms versus insoluble aggregates especially under stress conditions?4. Can the information gleaned for eIF3, eIF2 and NatA complex formation be used to direct the formation of translation factories for heterologous protein expression?