NEW / CURRENT: Systems modelling of a translational negative feedback loop: an in vivo toolkit to dissect ribosomal termination and mRNA surveillance

近年来，许多基因组序列已经完成，当然包括人类基因组。基因组中的每个基因都用来指导特定蛋白质的合成。正是蛋白质在细胞中起着功能性作用，例如作为催化剂加速细胞内的个别化学反应。基因中的信息被编码为碱基A、T、C和G的不同序列，在两个阶段的过程中被用来制造蛋白质。首先，基因信息以信使RNA （mRNA）的形式被复制到类似的碱基链中。信使rna是一种长链状分子，它被用作信息存储，指导由氨基酸链组成的蛋白质的组装，这一过程被称为翻译。氨基酸的精确序列（由mRNA碱基序列指导）决定了蛋白质的最终功能。氨基酸序列由mRNA序列定义，而mRNA序列又由基因序列定义，从而将基因与蛋白质联系起来。翻译过程是本研究的重点。在翻译过程中，被称为核糖体的小颗粒（本身由RNA和蛋白质组成）沿着mRNA移动，依次添加氨基酸，形成最终的蛋白质。这条生产线的过程根据mRNA中特定的碱基序列而停止（终止），导致完成的蛋白质的释放。终止对于确保所合成的蛋白质具有正确的长度至关重要。现在已经知道，在终止后，核糖体被引导回到mRNA的起点，有效地在mRNA链上循环利用它们。这使得翻译过程更有效率，但在mRNA生产线上产生非常复杂的核糖体流量。因此，核糖体流动的数学模型将与生化实验一起用于本研究，以帮助揭示控制翻译的机制。本文研究翻译终止有两个重要原因。首先，在许多人类遗传疾病中，受影响的基因（如囊性纤维化、杜氏肌营养不良）会发生突变，因为它在基因序列的早期含有一个额外的停止密码子。这有过早终止翻译的效果，导致缩短，无功能的蛋白质。人们对开发降低翻译终止准确性的药物越来越感兴趣。这将允许核糖体绕过早期终止密码子并到达自然终止密码子以生成正确长度的蛋白质。正如该提案所描述的那样，对终止的分子机制的研究可以提供关键的见解，直接用于开发治疗某些人类遗传疾病的药物。终止也很重要，因为与这个过程相关的是mRNA上核糖体的再循环。在信息末端终止后，核糖体主动返回到mRNA的起始处，从相同的模板合成新的蛋白质，形成一种环形核糖体赛跑轨迹；轨道的每一次循环都会产生一种新的蛋白质。人们对这种循环过程知之甚少，但它却是蛋白质合成效率的关键。通过了解回收是如何工作的，就有可能提高细胞中蛋白质合成的效率，这对于制造乙肝疫苗和胰岛素等药物至关重要。总之，翻译终止过程在每个细胞的基因表达中至关重要，因此具有基本的“纯粹”研究兴趣。然而，它也是理解如何在药物制造中重要的生物技术过程中有效地制造蛋白质的关键，也是治疗一系列极度衰弱的人类遗传疾病的药物的一个有吸引力的靶标。

Recent years have seen a host of genome sequences being completed, including of course the human genome. Each gene in a genome is used to direct the synthesis of a specific protein. It is the proteins that are the functional agents in a cell, for example acting as catalysts to speed individual chemical reactions within the cell. Information in the gene, coded as different sequences of the bases A, T, C and G, is used to make a protein in a two-stage process. First, the gene information is copied into a similar chain of bases in the form of a messenger RNA (mRNA). The mRNA, a long chain-like molecule, is then used as an information store to direct the assembly of a protein, consisting of a chain of amino acids, in a process called translation. The precise sequence of amino acids (directed by the mRNA base sequence) determines the eventual function of the protein. The amino acid sequence is defined by the mRNA sequence, which in turn is defined by the gene sequence, thus linking gene to protein. The process of translation forms the focus of this research proposal. During translation, small particles called ribosomes (themselves made of RNA and protein) travel along the mRNA, sequentially adding amino acids to make the final protein. This production line process is stopped (terminated) in response to a specific sequence of bases in the mRNA, causing the release of the completed protein. Termination is crucial for ensuring the protein made is of the correct length. It is now known that following termination, ribosomes are directed back to the beginning of the mRNA, effectively recycling them on the mRNA chain. This makes the translation process more efficient, but generates very complex ribosome traffic flow on the mRNA production line. For this reason, mathematical modelling of ribosome flow will be used in this research alongside the biochemical experimentation to help unravel the mechanisms by which translation is controlled. This proposal seeks to study translation termination for two important reasons. First, in many human genetic diseases, the affected gene (e.g cystic fibrosis, Duchenne muscular dystrophy) is mutated because it contains an additional stop codon early in the gene sequence. This has the effect of prematurely terminating translation, resulting in a shortened, non-functional protein. There is increasing interest in developing drugs that would make translation termination less accurate. This would allow the ribosome to bypass the early stop codon and reach the natural stop codon to make correct length protein. Research into the molecular mechanisms of termination, as this proposal describes, can provide crucial insight used directly in the development of drugs to treat some forms of human genetic disease. Termination is also important because associated with this process is the recycling of the ribosomes on the mRNA. After termination at the end of the message, ribosomes are actively returned to the beginning of the mRNA to make a new protein from the same template, forming a type of circular ribosomal race track; each circuit of the track results in a new protein being made. The recycling process is very poorly understood, and yet it is key to protein synthetic efficiency. By understanding how recycling works, it may be possible to boost the efficiency of protein synthesis in cells, which is crucial for the manufacture of drugs like hepatitis B vaccine and insulin, to name but two. In summary then, the process of translation termination is crucially important in the expression of genes in every cell, and thus has fundamental 'pure' research interest. It is however also a key to understand how proteins can be made efficiently in biotechnological processes important in drug manufacture, and is also an attractive target for drugs that can treat a range of extremely debilitating human genetic diseases.