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

通过协调 mRNA 定位优化异聚蛋白复合物的形成

• 批准号: 2449207
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2449207
• 关键词: Optimisation; heteromeric; protein; complex; formation

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?

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。获得的信息将应用于生产异源异聚复合物，这些复合物在酵母中具有重要的商业价值（例如血红蛋白和人促性腺激素）。更广泛地说，所学到的经验教训通常适用于使用酵母作为宿主生物体生产异源蛋白质。该学生项目的生物技术重点使其非常适合 BBSRC 的高级主题 2“应对战略挑战”。更具体地说，在该项目中，学生将研究 mRNA 到翻译工厂的协调本地化和翻译。要研究的 mRNA 编码异聚蛋白复合物的亚基，要解决的关键问题是所得蛋白复合物的折叠、组装和生物功能是否由于胞质翻译工厂中特定位点的生产而受到调节。酵母中的 eIF3、eIF2 和 NatA 复合物将用作示例蛋白质复合物，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，其中蛋白质合成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。这三个复合体都非常丰富且重要，因此它们在特定位点共翻译形成也就不足为奇了，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，然后将其应用于生物技术相关的异源蛋白质复合物的生产 - 评估将异源 mRNA 靶向蛋白质工厂是否会提高蛋白质复合物的生产力和产量。学生将接受多种不同方法的培训和分析作为该项目的一部分。基于活细胞 MS2 的成像技术和单分子荧光原位杂交将用于确定细胞中 mRNA 的位置。创新的荧光测定将用于研究每个 mRNA 的翻译位点，多核糖体分析将用于表征翻译效率。生化测定将用于研究可溶性或蛋白质聚集部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测定将用于剖析 eIF3 组装过程。将解决的问题是：1 . 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会改变以可溶活性形式存在的蛋白质复合物亚基与不溶性聚集体的比例，特别是在应激条件下？4。收集的 eIF3、eIF2 和 NatA 复合物形成信息能否用于指导异源蛋白表达翻译工厂的形成？