Mechanism of stringent translation initiation: a probe for its biological relevance

真核生物翻译起始是一个复杂的过程，涉及核糖体、mRNA、Met-tRNAiMet 和众多的真核细胞起始因子(EIF)。几十年来由那些使用 模式真核酵母酿酒酵母揭示了关键过程是形成 小核糖体P位点密码子-反密码子碱基配对。通过以下方式启用严格的启动 48S核糖体前起始复合体(PIC)严格在8月起始密码子形成，而 不包括在其他地点的启动。然而，有趣的是，许多非规范的Start站点被利用了 在某些生物学背景和疾病中，如癌症和神经退行性疾病。这个 人类基因组中非AUG起始位点的列表还远远没有完成，以及如何使用 因此，这些位点的蛋白质生产是否受到调控仍然是一个开放的问题 问题。 因此，这笔赠款的第一个目标是通过全基因组建立这样一个非8月起始位点的列表 对具有良好特征的癌症模型系统进行翻译分析，验证其中一些站点 并确定在癌症中驱动观察到的非AUG翻译调控的机制。 目的2研究5MP和Met-tRNAiMet腺苷N6-的作用机制。 苏氨酰氨基甲酰化(T6A)在控制非Aug翻译中的作用。如果是5MP，将会进行测试 在许多类型的癌症中发现的突变可以改变启动的准确性，从而影响患者的 预后。我们的初步研究表明，T6A位于Met-tRNAiMet反密码子的3‘端可以 专门歧视Gug和UUG开始密码子，而eIF1则更多 通用非Aug鉴别器。结合分子动力学模拟方法，我们将测试 如果最近发现的循环t6A起到区分作用，并确定 T6A和eIF1之间的合作或竞争促进了严格的启动和泄漏扫描 对转化性监管至关重要。 目3是研究已开发的起始密码子选择的一种不同机制 在平移水平的热冲击响应期间。这种机制是被发现的 通过翻译图谱分析与RNA结合缺陷的酵母eIF3i突变体 EIF3G亚基在高温下。工作假说假设，在高温下， 在AUG密码子上稳定的PIC形成需要额外的mRNA元件锚定 在其入口处启动核糖体。这些假定的信使核糖核酸元件位于下游 包括一个新的eIF3g结合基序5‘-GUCG-3’和一个下游的茎- 环，潜在地结合了40年代的进入部位侧，从而稳定了PIC形成。 该模型将使用酵母作为模型系统进行测试。

Eukaryotic translation initiation is a complex process involving the ribosome, mRNA, Met-tRNAiMet and numerous eukaryotic initiation factors (eIFs). Decades of studies driven by those using the model eukaryote yeast Saccharomyces cerevisiae revealed that the key process is the formation of codon-anticodon base pairing in the small ribosome P-site. Stringent initiation is enabled by formation of the 48S ribosomal pre-initiation complex (PIC) strictly at the AUG start codon while excluding initiation at other sites. Intriguingly, however, many non-canonical start sites are utilized in some biological contexts and diseases such as cancer and neurodegenerative disorders. The list of non-AUG start sites within the human genome is far from being complete and how the use of these sites and hence protein production from these sites are regulated remains an open question. Thus, Aim 1 of this grant is to make such a list of non-AUG start sites through genome-wide translation profiling of well characterized cancer model systems, verify some of these sites and determine the mechanism driving the observed non-AUG translational regulation in cancer. The Aim 2 is to study the mechanistic role of 5MP and Met-tRNAiMet adenosine N6- threonylcarbamoylation (t6A) in controlling non-AUG translation. It will be tested if 5MP mutations found in many types of cancer can alter initiation accuracy, thereby affecting patients' prognosis. Our preliminary studies suggested that t6A located 3' of Met-tRNAiMet anticodon can discriminate specifically against GUG and UUG start codons, in contrast to eIF1 being more universal non-AUG discriminator. Combining molecular dynamics simulation methods, we will test if the recently discovered cyclic t6A serves the discriminating role and determine how the cooperation or competition between t6A and eIF1 promotes stringent initiation and leaky scanning crucial for translational regulation. The Aim 3 is to study yet a distinct mechanism of start codon selection that is exploited during the heat shock response at the translational level. This mechanism was discovered through translational profiling of yeast eIF3i mutant defective in its interaction with RNA-binding eIF3g subunit at a high temperature. The working hypothesis assumes that, at a high temperature, stable PIC formation at the AUG codon requires additional mRNA elements anchoring the initiating ribosome at its entry site. These hypothetical mRNA elements are located downstream of the start codon and include a novel eIF3g-binding motif 5'-GUCG-3' and a downstream stem- loop that potentially binds the entry site-side of the 40S, thereby stabilizing the PIC formation. This model will tested using yeast as a model system.