Mechanism of action of codon pair bias

描述（由申请人提供）：由于氨基酸可以由多达六个密码子编码，因此存在许多不同的方式来编码任何特定的蛋白质。密码子的使用方式中存在偏见。一个众所周知的偏好是密码子偏好，简单地说，就是一些密码子比其他密码子使用得更多。不太为人所知的是，也存在“密码子对偏好”，使得一些密码子“偏好”与某些其他密码子相邻。这种密码子对偏好性与密码子偏好性是完全分开和独立的。最近，我们发现，当病毒被重新编码为具有不良密码子对偏好时，病毒被减毒，在极端情况下至无活力。似乎通过不良密码子对偏好性的病毒减毒可用于制备活的减毒疫苗。然而，虽然这个程序有效，但对于密码子对偏好性导致减毒的机制一无所知，这种知识的缺乏正在减缓病毒疫苗的工作。在这个建议中，我们将调查，为第一次，由坏密码子对偏好的衰减机制。这将在酵母中完成，我们最近已经证明了存在强烈的密码子对偏好效应。我们将使用两个密码子对去优化的合成酵母基因dHIS 3和dLYS 2来确认密码子对偏好在酵母中的影响。我们将使用多核糖体分析和核糖体密度图来测试坏密码子对偏好性减缓翻译的想法。我们将通过关闭蛋白酶体降解以及使用其他方法来测试坏密码子对偏好导致不准确翻译和蛋白质降解的想法。我们已经选择了酵母突变体，这些突变体对密码子对偏好的敏感性明显低于野生型，我们将描述这些突变体的特征并鉴定突变基因。最后，我们将对（最初）22,000种不同的HIS 3编码进行高通量研究，以将特定编码与基因功能的强度相关联。我们的长期目标是了解通过不良密码子对偏好的减毒机制，以促进活的减毒病毒疫苗的开发，并促进基因表达的调节。 公共卫生相关性：制造抗病毒疫苗的一种方法是使病毒突变以削弱它，然后使用这种减弱的病毒作为疫苗（例如，FluMist，一种活流感疫苗）。然而，这样做有许多困难。最近，我们发现了一种新的方法，密码子对去优化，为此目的。虽然这个方法有效，但我们不知道它为什么有效。在这里，我们将研究通过密码子对去优化的减毒机制。

DESCRIPTION (provided by applicant): Because an amino acid can be encoded by as many as six codons, there are many different ways to encode any particular protein. Biases exist in the way codons are used. One well- known bias is the codon bias, which is simply that some codons are used more than others. Less well-known is that there is also a "codon pair bias", such that some codons "prefer" to be adjacent to certain other codons. This codon pair bias is completely separate and independent from the codon bias. Recently, we have found that when viruses are re-coded to have a bad codon pair bias, the viruses are attenuated, in extreme cases to inviability. It appears that attenuation of a virus via a bad codon pair bias can be used to make a live, attenuated vaccine. However, while the procedure works, nothing whatever is known about the mechanim by which codon pair bias causes attenuation, and this lack of knowledge is slowing the work with viral vaccines. In this proposal, we will investigate, for the first time, the mechanism of attenuation by bad codon pair bias. This will be done in yeast, where we have recently shown there are strong codon pair bias effects. We will confirm the effects of codon pair bias in yeast using two codon pair de-optimized synthetic yeast genes, dHIS3 and dLYS2. We will test the idea that bad codon pair bias slows translation using polysome profiling and ribosome density mapping. We will test the idea that bad codon pair bias causes inaccurate translation and protein degradation by turning off proteasomal degradation, and also using other methods. We have already selected yeast mutants that are apparently less sensitive than wild-type to codon pair bias, and we will characterize these mutants and identify the mutant genes. Finally we will do a high- throughput study of (initially) 22,000 different encodings of HIS3 to correlate particular encodings with the strength of gene function. Our long term goal is to understand the mechanism of attenuation by bad codon pair bias to facilitate the development of live, attenuated viral vaccines, and also to facilitate the tuning of gene expression. PUBLIC HEALTH RELEVANCE: One way to make an anti-viral vaccine is to mutate the virus to weaken it, then use this weakened virus as a vaccine (e.g., FluMist, a live Flu vaccine). However there are many difficulties in doing this. Recently we have found a new method, codon pair de-optimization, for this purpose. Although the method works, we do not know why it works. Here, we will study the mechanism of attenuation by codon pair de-optimization.