Structure and Function of Plant Protein Synthesis Initiation Factors

真核生物蛋白质合成的启动是一个复杂的过程，至少需要8-10个已知的启动因子。PI实验室的长期目标是阐明植物中这一过程的详细分子描述。为此，他们分离和克隆了植物起始因子，以确定它们的生化性质和结构，并最终绘制出它们之间的物理和功能相互作用，mRNA和核糖体。本研究的总体目标是了解两种形式的eIF4F在植物翻译启动中的作用。我们将探讨三个具体问题。首先，eIF4F和eIF(Iso)4F复合体的分子组成如何影响信使核糖核酸翻译的特异性，以及信使核糖核酸(5‘非编码区、3’非编码区或编码区)的什么特征影响eIF4F和eIF(Iso)4F的区分？植物有第二种新的eIF4F形式，eIF(Iso)4F。一些mRNAs在体外被eIF(Iso)4F翻译得很差，而另一些则不显示出对eIF4F或eIF(Iso)4F的偏好。用混合亚基复合体(eIF4G/eIF(Iso)4E和eif(Iso)4G/eif4E)确定具有翻译特异性的eIF4F和eIF(Iso)4F的亚基(S)。此外，eIF4F和eIF(Iso)4F区分所需的mRNA5‘、3’和/或编码区部分将使用嵌合mRNAs来确定。第二，eIF(Iso)4F对于植物生存是多余的还是必要的？遗传方法将被用来确定敲除或基因沉默的eIF(Iso)4F亚基、eIF(Iso)4E和eIF(Iso)4G是否产生任何明显的表型和/或对存活的植物是必需的。最后，eIF4G的结合如何影响eIF4E的结构和对mRNA的亲和力？从eIF4G中提取的含有eIF4E结合位点的多肽将被用来制成络合物，以研究eIF4E结合时结构的变化。将进行进一步的研究，以衡量与eIF4E相比，该复合体的mRNA亲和力的变化。蛋白质合成是所有细胞必不可少的分子过程。了解因子、核糖体和信使核糖核酸之间的相互作用对于整合这一过程及其与其他细胞过程的调控是必要的。很明显，在所有高等生物中，启动蛋白质合成的某些方面非常相似，但有一些根本的不同。EIF4F同工酶的存在表明，植物可能保留了更古老的eIF4F复合体，或者为植物的某些特定目的或功能进化了第二个复合体。清楚地了解植物是如何制造蛋白质的，将有助于开发营养价值更高的植物或作为生产蛋白质药物的活工厂。该项目得到了生物科学局分子和细胞生物科学部的基因表达生物化学计划和分子生物化学计划的支持。

The initiation of protein synthesis in all eukaryotes is an intricate process requiring at least 8-10 known initiation factors. The long term goal of the PI's laboratory is to elucidate a detailed molecular description of this process in plants. Towards this end they have isolated and cloned plant initiation factors to determine their biochemical properties and structures, and ultimately to map their physical and functional interactions with each other, mRNA and ribosomes. The overall objective of this research is to obtain an understanding of the function of two forms of eIF4F in plant translation initiation. Three specific questions will be explored. First, how does the molecular composition of eIF4F and eIF(iso)4F complexes affect specificity of mRNA translation, and what features of a mRNA (5' UTR, 3' UTR or coding region) affect discrimination by eIF4F and eIF(iso)4F? Plants have a second novel form of eIF4F, eIF(iso)4F. Some mRNAs are translated poorly in vitro by eIF(iso)4F, whereas others do not show a preference for eIF4F or eIF(iso)4F. The subunit(s) of eIF4F and eIF(iso)4F that confer specificity of mRNA translation will be determined using mixed subunit complexes (eIF4G/eIF(iso)4E and eIF(iso)4G/eIF4E). In addition, the portions of the mRNA (5' UTR, 3' UTR and/or coding region) that are necessary for discrimination by eIF4F and eIF(iso)4F will be determined using chimeric mRNAs. Second, is eIF(iso)4F redundant or necessary for plant survival? Genetic methods will be used to determine whether knockouts or gene silenced eIF(iso)4F subunits, eIF(iso)4E, and eIF(iso)4G produce any obvious phenotypes and/or are necessary for viable plants. Finally, how does the binding of eIF4G affect eIF4E structure and affinity for mRNA? A peptide derived from eIF4G that contains the eIF4E binding site will be used to make a complex to study the changes in eIF4E structure upon its binding. Further studies will be carried out to measure changes in mRNA affinity for the complex compared to eIF4E.Protein synthesis is an essential molecular process for all cells. An understanding of the interactions of the factors, ribosomes and mRNA is necessary to integrate this process and its regulation with other cellular processes. It is clear that some aspects of the initiation of protein synthesis are very similar among all higher organisms, but that there are some fundamental differences. The presence of the isozyme form of eIF4F suggests that plants may have retained a more ancient form of the eIF4F complex or evolved a second complex for some specific purpose or function in plants. A clear understanding of how plants manufacture their proteins will help in the development of plants with higher nutritive value or as living factories for the production of protein pharmaceuticals.This project is supported by the Biochemistry of Gene Expression Program and the Molecular Biochemistry Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.