Structure of Plant Protein Synthesis Initiation Factors

布朗宁9406601本研究的具体目标是：1）确定小麦胚eIF-（iso）4F和EIF-4F亚基的功能结构域。 eIF-（iso）4F亚基已在E.大肠杆菌中，并重组以形成酶活性复合物。 将在eIF-（iso）4F和eIF-4F的亚基中进行突变，并在体外麦胚翻译系统和几种测量部分反应的体外测定中进行测试，即，ATP水解、RNA解旋、与mG帽交联以及mRNA与40 S核糖体亚基结合。 2)完成了小麦eIF-4 B的部分cDNA序列，并在E.杆菌 通过RACE-PCR、反向PCR或基因组测序获得eIF-4 B cDNA的缺失部分（约100-200个核苷酸）。 该cDNA将在E.大肠杆菌中的酶活性和在上述测定中测定的酶活性（具体目的1）。 3)完成拟南芥eIF-4F、eIF-（iso）4F的p28和eIF-4 B亚基的cDNA序列，并在E.杆菌 我们将继续使用小麦因子和小麦cDNA序列的抗体进行筛选。 将小麦cDNA的不同区域用于制备用于筛选的探针。 拟南芥因子的cDNA将在大肠杆菌中表达。 将在上述测定中测量表达的拟南芥属因子的活性。 这一目标的完成将为今后在体内探索抑制因子表达的控制和植物蛋白质合成的调节奠定基础。 这个项目的长期目标是阐明植物蛋白质合成起始的步骤。 具体来说，我们要确定的结构方面的植物蛋白质合成起始因子（eIFs）的相互作用的因素是很重要的，与mRNA和ATP，和mRNA的结合40 S核糖体亚基。 本研究的主要目的是：1）利用突变技术确定eIF-（iso）4F和eIF-4F亚基的功能结构域。 2)完成了小麦eIF-4 B和eIF-4F的p220基因的cDNA序列，并在E. 杆菌 3)完成拟南芥eIF-（iso）4F、eIF-4F和eIF-4 B亚基的cDNA序列，并在E.杆菌 这项研究的结果将增强我们对这些重要蛋白质的理解，并提供有关其结构和功能的宝贵信息。 ***

Browning 9406601 The specific objectives of this proposal are to: 1) Determine the functional domains of the subunits of wheat germ eIF-(iso)4F and EIF-4F. The subunits of eIF-(iso)4F have been expressed in E. coli and recombined to form an enzymatically active complex. Mutations will be made in the subunits of eIF-(iso)4F and eIF-4F and tested in an in vitro wheat germ translation system and several in vitro assays that measure partial reactions, i.e., ATP hydrolysis, RNA unwinding, cross-linking to the mG cap and binding of mRNA to 40S ribosomal subunits. 2) Complete the partial cDNA sequence of wheat eIF-4B and express this protein in E. coli. The missing portion (~100-200 nucleotides) of the cDNA for eIF-4B will be obtained by RACE-PCR, inverse PCR or genomic sequencing. The cDNA will be expressed in E. coli and the enzymatic activity determined in the assays described above (Specific Objective 1). 3) Complete the cDNA sequence for the subunits of Arabidopsis eIF-4F, p28 of eIF- (iso)4F and eIF-4B and express these proteins in E. coli. We will continue to screen using antibodies to wheat factors and wheat cDNA sequences. Different regions of the wheat cDNAs will be used to prepare probes for screening. The cDNAs for Arabidopsis factors will be expressed in E coli. The activity of the expressed Arabidopsis factors will be measured in the assays described above. The completion of this goal will set the stage for future work in vivo exploring the control of expression of the inhibition factors and regulation of protein synthesis in plants. %%% The long term goal of this project is to elucidate the steps in the initiation of protein synthesis in plants. Specifically we want to determine the structural aspects of plant protein synthesis initiation factors (eIFs) that are important for the interaction of the factors with each other, with mRNA and ATP, and for the binding of mRNA to 40S ribosomal subunits. The project goals are to: 1) Determine the fun ctional domains of the subunits of eIF-(iso)4F and eIF-4F using mutagenesis. 2) Complete the cDNA sequence of wheat eIF-4B and p220 of wheat eIF-4F and express these proteins in E. coli. 3) Complete the cDNA sequence for the subunits of Arabidopsis eIF-(iso)4F and eIF-4F and eIF-4B and express these proteins in E. coli. The results of this research will enhance our understanding of these important proteins and provide valuable information about their structure and function. ***