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Analysis of the mechanism for coupled translation in cellular and virus mRNAs

细胞和病毒 mRNA 耦合翻译机制分析

基本信息

批准号：
BB/I022880/1
负责人：
Andrew Easton
金额：
$ 58.05万
依托单位：
University of Warwick
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2011
资助国家：
英国
起止时间：
2011 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FI022880%2F1
关键词：
Analysis mechanism coupled translation cellular

项目摘要

Translation is the final stage of the most fundamental process in biology in which the genetic material of the organism is turned into proteins. During the process of translation; ribosomes (which are the cellular machines that build proteins) make the new protein by following a plan written in a molecule called mRNA. mRNA is a single stranded molecule made up from 4 different components (called nucleotides) which act like letters in our alphabet to make a code. Scientists have been studying the way the ribosome machine works in eukaryotes (higher organisms) for many years and thought that all proteins are made in the same way. Viruses are a group of microscopic organisms which infect and cause diseases in organisms. During the infection process they must use the infected cell's apparatus including ribosomes to make more new viruses. Viruses have been forced to exploit all of the cell systems to survive and multiply faster before the infected cell can destroy it. Importantly, all strategies used by the virus must be compatible with the workings of the cell to function. Interestingly, by studying these new strategies Scientists have seen that not only, do the viruses use them, but the cells also do. We are studying a virus called respiratory syncytial virus (RSV) which will have likely infected most individuals a child and will have a good chance of infected them again as adults. The RSV virus also uses a novel mechanism to express a protein called M2-2. Here instead of just making one protein from a piece of mRNA two proteins are made. We are interested in working out how this is possible. We have shown that in order to make the second protein: M2-2 the first protein M2-1 must be made. This means the ribosomes must go in reverse as the coding sequence for the second protein overlaps with the coding sequence of the first. This was an important finding as ribosomes had not previously been shown to have this ability. Important regions within this mRNA have been discovered that allow the second protein to be made. These regions appear to be present throughout the coding sequence of M2-1. If we change these regions by introducing mutations or make deletions the amounts of the second protein made are reduced, in some cases completely, indicating their importance. These sections contain a lot of secondary structure. Secondary structure occurs where the nucleotides in different parts of the mRNA molecule can interact together, which can be thought of as like fitting two pieces of a jig-saw together. As more pieces are added the structure gets stronger. We are going to use the latest techniques to capture and pinpoint where the ribosome is on the M2-1 RNA. If the ribosome is in one location more than by random chance this will indicate the location where the ribosome has paused. This could 1) allow the ribosome to effectively think and change its mind. So instead of doing what it should and following a signal to stop and move away it re-starts and makes a new protein and or 2) The secondary structures inhibit ribosome movements so that gaps appear on the mRNA molecule so a ribosome stopping can also move back and not be blocked. We are also interested in finding out if other proteins assist in this process. We aim to fish out these proteins using our M2-1 RNA as bait. We have also discovered for the first time that our genome contains examples of mRNA that carry out this process. We have at least 5 examples to investigate further and highlights the universal use of this mechanism.

翻译是生物学中最基本的过程的最后阶段，在这个过程中，生物体的遗传物质转化为蛋白质。在翻译过程中，核糖体（构建蛋白质的细胞机器）通过遵循一种称为mRNA的分子中编写的计划来制造新蛋白质。mRNA是一种单链分子，由4种不同的成分（称为核苷酸）组成，它们就像我们字母表中的字母一样构成代码。科学家们多年来一直在研究真核生物（高等生物）中核糖体机器的工作方式，并认为所有蛋白质都是以同样的方式产生的。病毒是一组微生物，它们感染并引起生物体的疾病。在感染过程中，它们必须利用被感染细胞的器官（包括核糖体）来制造更多的新病毒。病毒不得不利用所有的细胞系统在被感染的细胞摧毁之前生存和繁殖得更快。重要的是，病毒使用的所有策略必须与细胞的运作兼容。有趣的是，通过研究这些新策略，科学家们发现，不仅病毒使用它们，而且细胞也使用它们。我们正在研究一种叫做呼吸道合胞病毒（RSV）的病毒，这种病毒很可能在儿童时期感染大多数人，并且在成年后很有可能再次感染。RSV病毒还使用一种新的机制来表达一种名为M2-2的蛋白质。在这里，不是从一段mRNA中合成一种蛋白质，而是合成两种蛋白质。我们有兴趣弄清楚这是如何可能的。我们已经证明，为了制造第二种蛋白质：M2-2，必须制造第一种蛋白质M2-1。这意味着核糖体必须反向运动，因为第二种蛋白质的编码序列与第一种蛋白质的编码序列重叠。这是一个重要的发现，因为核糖体以前没有被证明具有这种能力。在这个mRNA中的重要区域已经被发现，允许第二个蛋白质被制造。这些区域似乎存在于M2-1的整个编码序列中。如果我们通过引入突变或进行缺失来改变这些区域，则所产生的第二种蛋白质的量减少，在某些情况下完全减少，这表明它们的重要性。这些部分含有大量的二级结构。二级结构发生在mRNA分子不同部分的核苷酸可以相互作用的地方，这可以被认为是将两块拼图拼在一起。随着更多的部件被添加，结构变得更坚固。我们将使用最新的技术来捕捉和精确定位核糖体在M2-1 RNA上的位置。如果核糖体在一个位置上多于随机的机会，这将指示核糖体已经暂停的位置。这可以1）允许核糖体有效地思考和改变它的想法。因此，它没有做它应该做的事情，而是按照一个信号停止并离开，它重新开始并制造一个新的蛋白质，或者2）二级结构抑制核糖体运动，这样mRNA分子上就会出现缺口，这样核糖体停止也可以向后移动，而不会被阻止。我们也有兴趣了解其他蛋白质是否有助于这一过程。我们的目标是用我们的M2-1 RNA作为诱饵钓出这些蛋白质。我们还首次发现，我们的基因组中含有执行这一过程的mRNA。我们至少有5个例子来进一步研究，并强调了这种机制的普遍使用。