Structure of Plant Protein Synthesis Initiation Factors

本课题的研究目标是：1)克隆小麦胚芽真核起始因子(eIF)-4F的两个亚基（p220和p26）及其同工酶形式eIF-(iso)4F的两个亚基（p82和p28）的cdna并进行测序。2)通过比较p26和p28多肽的序列以及与其他细胞（酵母、小鼠、人）的m7G帽结合蛋白的序列，确定p26和p28的m7G帽结合位点。此外，我们将对p220和p82的序列进行比较，以确定这些肽的ATP结合位点、mRNA结合位点和RNA解旋酶结构域。3)克隆eIF-4A和eIF-4B的cdna序列，并将这些序列与从其他真核细胞中获得的序列进行比较，并尝试鉴定功能域。4)利用小麦cDNA克隆确定拟南芥中eIF-4A、eIF-4B以及eIF-4F和eIF-iso)4F亚基基因的数量、位置和序列。5)利用小麦的eIF-4A cDNA克隆；eIF- 4b以及eIF-4F和eIF-(iso)4F的亚基，以确定这些基因在植物发育不同阶段的表达水平。为了实现这些目标，将使用各种方法筛选小麦cDNA表达文库。针对起始因子的抗体是可用的，以及一些肽序列的知识，这些知识将用于设计寡核苷酸探针。DNA测序、Southern和Northern blot分析以及mRNA的定量将通过已建立的技术进行。目前尚不清楚eIF-4A、eIF-4B和eIF-4F的哪些结构特征对其功能有重要作用。这些目标的完成将为这些多肽的结构特征提供有价值的信息，这些多肽对于识别mRNA的5' m7G帽，与mRNA结合，atp依赖性RNA解旋酶活性以及mRNA与核糖体结合非常重要。

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.