Structure of Plant Protein Synthesis Initiation Factors

本研究建议的项目目标是： 1)克隆和测序两个亚基的cDNA， 麦胚真核起始因子（eIF）-4F（p220和 p26）及其同工酶形式eIF-（iso）4F的两个亚基 (p82 p28）。 2)通过以下方法鉴定p26和p28的m7 G帽结合位点： 将这些多肽的序列相互比较 来自其它细胞的一种至m7 G帽结合蛋白（酵母， 小鼠、人）。 此外，还比较了 p220和p82将被用于鉴定ATP 结合位点、mRNA结合和RNA解旋酶结构域 缩氨酸 3)克隆eIF-4A和eIF-4 B的cDNA序列， 将这些序列与从其他来源获得的序列进行比较。 真核细胞，并试图确定功能 域. 4)为了使用小麦cDNA克隆来确定数量， 拟南芥中eIF-4A基因的位置和序列， eIF-4 B以及eIF-4F和eIF-4F的亚基。 5)为了使用eIF-4A、eIF-4 B和eIF-4 B的小麦cDNA克隆， eIF-4F和eIF-（iso）4F的亚基，以确定eIF-4F和eIF-（iso）4F的水平。 这些基因在植物的各个阶段的表达 发展 为了实现这些目标，各种方法来筛选小麦 将使用cDNA表达文库。 抗体与 启动因素是可用的，以及一些知识 这些肽序列将被用来设计 寡核苷酸探针。 DNA测序，Southern和 将进行北方印迹分析和mRNA定量。 通过已建立的技术。 不知道是什么 eIF-4A、eIF-4 B和eIF-4F的结构特征是 重要的是他们的功能。 实现这些目标将 提供有关结构的宝贵信息 这些多肽的特征对于 识别mRNA的5 ′ m7 G帽，用于结合mRNA， 对于ATP依赖性RNA解旋酶活性和对于 mRNA到核糖体。

The project goals of this research proposal are: 1) to clone and sequence the cDNAs for the two subunits of wheat germ eukaryotic initiation factor (eIF)-4F (p220 and p26) and the two subunits of its isozyme form, eIF-(iso)4F (p82 and p28). 2) to identify the m7G cap binding site of p26 and p28 by comparing the sequences of these polypeptides to each other an to m7G cap binding proteins from other cells (yeast, mouse, human). In addition, a comparison of the sequences of p220 and p82 will be made in an effort to identify the ATP binding site, mRNA binding and RNA helicase domains of these peptides. 3) to clone an sequence the cDNAs for eIF-4A and eIF-4B, to compare these sequences to those of obtained from other eukaryotic cells, and to attempt to identify functional domains. 4) to use the wheat cDNA clones to determine the number, location and sequence of the genes in Arabidopsis for eIF-4A, eIF-4B and the subunits of eIF-4F and eIF-iso)4F. 5) to use the wheat cDNA clones for eIF-4A, eIF-4B and the subunits of eIF-4F and eIF-(iso)4F to determine the level of expression of these genes during various stages of plant development. To achieve these goals, various methods to screen a wheat cDNA expression library will be used. Antibodies to the initiation factors are available, as well as some knowledge of peptide sequences which will be used to design oligonucleotide probes. DNA sequencing, Southern and Northern blot analysis and quantitation of mRNA will be carried out by established techniques. It is not known what structural features of eIF-4A, eIF-4B and eIF-4F are important for their function. Completion of these goals will provide valuable information with regard to the structural features of these polypeptides that are important for the recognition of the 5' m7G cap of mRNA, for binding to mRNA, for ATP-dependent RNA helicase activity and for binding of mRNA to ribosomes.