Mechanism of action of codon pair bias

描述(申请人提供)：因为一个氨基酸可以由多达六个密码子编码，所以有许多不同的方法来编码任何特定的蛋白质。密码子的使用方式存在偏见。一种众所周知的偏向是密码子偏向，它简单地说就是某些密码子比其他密码子使用得更多。不太为人所知的是，还有一种“密码子对偏向”，即一些密码子“更喜欢”与某些其他密码子相邻。这种密码子对偏向是完全独立于密码子偏向的。最近，我们发现，当病毒被重新编码为具有糟糕的密码子对偏向时，病毒会减弱，在极端情况下会丧失生存能力。看来，通过错误的密码子对偏向对病毒的减毒作用可以用来制造活的减毒疫苗。然而，尽管这一过程有效，但对密码子偏向导致衰减的机制知之甚少，这种知识的缺乏正在减缓病毒疫苗的工作。在这项提案中，我们将首次研究不良密码子对偏差对衰减的机制。这将在酵母中完成，我们最近证明了酵母中有很强的密码子对偏向效应。我们将使用两个密码子对去优化的合成酵母基因dHIS3和dLYS2来证实密码子对偏差在酵母中的影响。我们将使用多聚体图谱和核糖体密度图谱来测试不良密码子对偏见会减缓翻译速度的想法。我们将通过关闭蛋白酶体降解以及使用其他方法来测试不良密码子对偏见导致翻译不准确和蛋白质降解的想法。我们已经选择了对密码子对偏差明显不像野生型敏感的酵母突变株，我们将对这些突变株进行特征分析并鉴定突变基因。最后，我们将对(最初)22,000个不同的HIS3编码进行高通量研究，以将特定的编码与基因功能的强度相关联。我们的长期目标是了解不良密码子对偏差的衰减机制，以促进活的、减毒的病毒疫苗的开发，并促进基因表达的调节。 与公共卫生相关：制造抗病毒疫苗的一种方法是对病毒进行变异以削弱它，然后将这种减弱的病毒用作疫苗(例如，流感活疫苗FluMist)。然而，要做到这一点有很多困难。最近，我们发现了一种新的方法，密码子对去优化，以达到这一目的。虽然这种方法奏效了，但我们不知道它为什么奏效。在这里，我们将研究密码子对去优化的衰减机制。

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.