Structure of Plant Protein Synthesis Initiation Factors

布朗宁9406601本提案的具体目标是：1)确定小麦胚芽eIF-(Iso)4F和eIF-4F亚基的功能域。EIF-(Iso)4F的亚基已在大肠杆菌中表达，并重组形成具有酶活性的复合体。将在eIF-(Iso)4F和eIF-4F的亚基中进行突变，并在体外小麦胚芽翻译系统和几种衡量部分反应的体外检测中进行测试，即ATP水解、RNA解离、与MG帽的交联以及mRNA与40S核糖体亚基的结合。2)完成小麦eIF-4B基因的部分序列，并在大肠杆菌中表达该蛋白。EIF-4B基因缺失的部分(~100-200个核苷酸)将通过RACE-PCR、反向PCR或基因组测序获得。该cDNA将在大肠杆菌中表达，并在上述检测中测定酶活性(特定目标1)。3)完成了拟南芥eIF-4F、eIF-(Iso)4F和eIF-4B亚基的全长cDNA序列，并在大肠杆菌中进行了表达。我们将继续使用小麦因子抗体和小麦cdna序列进行筛选。小麦不同区域的cDNA将被用来准备用于筛选的探针。拟南芥因子的cDNA将在大肠杆菌中表达。表达的拟南芥因子的活性将在上述检测中被测量。这一目标的完成将为今后在体内探索抑制因子的表达调控和植物蛋白质合成的调控奠定基础。这个项目的长期目标是阐明启动植物蛋白质合成的步骤。具体地说，我们想要确定植物蛋白质合成起始因子(EIF)的结构方面，这些结构方面对于这些因子之间的相互作用、与mRNA和ATP的相互作用以及对mRNA与40S核糖体亚基的结合都是重要的。该项目的目标是：1)利用诱变技术确定eIF-(Iso)4F和eIF-4F亚单位的功能结构域。2)完成了小麦eIF-4B和小麦eIF-4F的p220基因的全序列，并在大肠杆菌中进行了表达。3)完成了拟南芥eIF-(Iso)4F、eIF-4F和eIF-4B亚基的基因序列，并在大肠杆菌中进行了表达。这项研究的结果将加深我们对这些重要蛋白质的了解，并提供关于它们结构和功能的有价值的信息。***

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. ***