REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITITATION

蛋白质合成在生长和分化中起核心作用， 对变化的响应以及电池的总体能量利用。病毒 利用宿主的翻译机器，宿主细胞已经进化出 复杂的抗病毒反应。蛋白质合成的失调导致 恶性转化这个项目旨在了解率- 蛋白质合成的限制步骤，mRNA向核糖体的募集， 在那里发生了大量的翻译控制。这个过程中的两个关键因素 是eIF 4 E，mRNA帽结合蛋白，和eIF 4G，一种连接 40 S核糖体、eIF 4 E和RNA解旋酶eIF 4A。的作用 eIF 4启动因子将通过以下问题进行研究 问题：1. eIF 4因子如何参与mRNA的募集 到核糖体上我们将捕获起始复合物的中间体 使用烯丙基寡核苷酸组装，过表达eIF 4G和蛋白酶- 在培养细胞中测定致癌表型的耐药变异体试验 eIF 4G特定功能结构域的作用，并表征eIF 4 E 梭与m7 G-和m3（2，2，7）G-结合的关系 上限eIF 4 E的磷酸化如何影响蛋白质合成？我们 将在CREF细胞中磷酸化位点周围表达eIF 4 E变体 为了确定表型，检查eIF 4 E的特征， 通过PKCzeta磷酸化，确定磷酸化的性质， eIF 4 E通过生物物理学方法，并测试eIF 4 E是否参与了一个 磷酸化周期启动。什么生理因素 确定细胞内水平和活性的eIF 4G？我们将研究 eIF 4G合成、降解和稳态水平作为几个功能 参数以及温度依赖性构象变化， 重组eIF 4G的生物物理和生物化学。IRES是如何 eIF 4G mRNA直接内部启动？我们将划定 eIF 4G IRES，确定其二级结构，表征其翻译 并试图寻找其在体外应用的条件。和 最后，eIF 4 E基因的结构是什么？我们将完成 确定基因结构，定义启动子，并试图 鉴定顺式作用调节元件。

Protein synthesis plays a central role in growth and differentiation, response to change, and overall energy utilization of the cell. Viruses utilize the host translational machinery, and host cells have evolved complex antiviral responses. Deregulation of protein synthesis leads to malignant transformation. This project seeks to understand the rate- limiting step of protein synthesis, recruitment of mRNA to the ribosome, where much of translational control occurs. Two key factors in this process are eIF4E, the mRNA cap-binding protein, and eIF4G, a protein which links together the 40S ribosome, eIF4E, and the RNA helicase eIF4A. The roles of the eIF4 initiation factors will be studied by asking the following questions: 1. How do the eIF4 factors participate in recruitment of mRNA to the ribosome? We will arrest of intermediates of initiation complex assembly using allyloligonucleotides, overexpress eIF4G and a protease- resistant variant in cultured cells to determine oncogenic phenotype, test the effect of specific functional domains of eIF4G, and characterize eIF4E from C. elegans with respect to binding to m7G- and m3(2,2,7) G-containing caps. How does the phosphorylation of eIF4E affect protein synthesis? We will express in CREF cells eIF4E variants around the phosphorylation site to determine phenotype, examine the characteristics of eIF4E phosphorylation by PKCzeta, determine the properties of phosphorylated eIF4E by biophysical methods, and test whether eIF4E participates in a phosphorylation cycle during initiation. What physiological factors determine the intracellular levels and activity of eIF4G? We will study eIF4G synthesis, degradation and steady-state levels as a function several parameters as well as temperature-dependent conformational changes in recombinant eIF4G by biophysical and biochemical. How does the IRES of eIF4G mRNA direct internal initiation? We will delineate the borders of the eIF4G IRES, determine its secondary structure, characterize its translation in vivo, and attempt to find conditions for its utilization in vitro. And finally, what is the structure of the eIF4E gene? We will complete determination of the gene structure, define the promoter, and attempt to identify cis-acting regulatory elements.