REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITIATION

真核生物中蛋白质合成的起始是 是通过一系列最复杂的生化反应完成的 已知反应。了解这一过程对于 了解蛋白质合成的速度是如何被调节的， 许多病毒的细胞毒作用，抗病毒的作用 蛋白质干扰素与基因的终极表达 信息。这项计划的长远目标有两个- 折叠：了解各种蛋白质的生化作用(ELF- 4个基团启动因子)参与信使RNA的进入 进入启蒙过程，并对启蒙因素负责 对于启动因子的活性和表达水平 EIF-4E(帽结合蛋白)。对于第一个目标，积极的 EIF-4E的地点将通过两种技术相结合来确定： 将合成一组新的光亲和标记并用于 标记eIF-4E的m7GTP结合位点。第二，改变形式的 EIF-4E将在体外通过定点突变产生。这个 EIF-4A、-4B、-4E和-4F在不同起爆方式上的定位 复合体将被确定。帽状结构的相互作用， 在m7GTP的mRNA和光亲和衍生物中，都有 将调查eIF-4组因素。最后，获得了一种新的 将对eIF-4F的p220部分进行克隆和测序。对于 第二个目标是，eIF-4E的磷酸化作用将是 检查过了。这将在体外通过使用 特异性激酶和eIF-4E形式的无细胞合成 缺乏磷酸化位点，由定点产生 诱变。它也将在体内进行研究，通过相互关联 磷酸化与蛋白质合成速率和使用瞬变 含有诱变形式的eIF-4E基因的表达载体。 最后，各种形式的eIF-4E mRNA的结构将 进行检测，这种蛋白质的基因将被克隆和 部分测序。 R0GM33804 具有以下特征的选定功能属性 将对细胞色素c进行研究，并可能赋予 这种具有新功能特性的蛋白质将被探索 通过建造一系列专门设计的 突变体如下：(1)推测的结晶学 底物结合部位的鉴定 将通过检查Ser-82变种的效果来评估 有关小分子物质对其氧化还原性能的影响 蛋白质。(2)轴向配基在决定 细胞色素功能将通过对Met-80突变体的分析来研究。 (3)第38位和第82位突变的改变机制 碱性转变将通过pH跳跃实验进行研究，EPR 光谱学和静电学计算。(4)高野 细胞色素c氧化还原相互转化的Dickerson模型将是 通过对最近构建的Thr-78突变体的研究进行评估。这 残基对Takano-Dickerson模型至关重要，因为它是氢- 与一个关键的、内部结合的水分子结合，这是关键的 他们的提议。(5)丙酸亚铁血红素-7的调节作用 蛋白质的还原潜力将通过以下方式进行研究 考虑一个Tyr-48突变体。TYR-48/Arg-的可能性分析 在这方面也将考虑38个双重突变体。(6) 将进一步分析Phe-82的多重作用 通过对在这个位置构建的几个新突变体的评估。 (7)细胞色素物种差异的来源将是 通过环的构造和特征来考虑 转化酵母异L细胞色素c的插入/缺失突变体 形成在大小上更接近两个原核细胞色素的形式。(8) 选择性突变对电子转移动力学的影响 对生理氧化还原伙伴蛋白进行了研究。(9)A类 一系列光谱技术(核磁共振、CD/MCD和时间分辨 荧光光谱学)将应用于选定的突变体 由它们观察到的特性决定。

Initiation of protein synthesis in eukaryotic organisms is accomplished through one of the most complex series of biochemical reaction known. Knowledge of this process is important for an understanding of how the rate of protein synthesis is regulated, the cytotoxic effect of many viruses, the action of the antiviral protein interferon, and the ultimate expression of genetic information. The long-term objectives of this project are two- fold: to understand the biochemical roles of various proteins (elF- 4 group initiation factors) involved in the entry of messenger RNA into the initiation process, and to initiation factors responsible for the activity and level of expression of the initiation factor eIF-4E (cap-binding protein). For the first objective, the active site of eIF-4E will be determined by a combination two techniques: a set of new photoaffinity labels will be synthesized and used to label the m7GTP-binding site of eIF-4E. Second, altered forms of eIF-4E will be produced in vitro by site-directed mutagenesis. The location of eIF-4A, -4B, -4E and -4F on various initiation complexes will be determined. The interaction of cap structures, both in mRNA and in photoaffinity derivatives of m7GTP, with the eIF-4 group factors will be investigated. Finally, the cDNA for the p220 component of eIF-4F will be cloned and sequenced. For the second objective, the effect of phosphorylation of eIF-4E will be examined. This will be studied in vitro through the use of specific kinase and by cell-free synthesis of forms of eIF-4E lacking a phosphorylation site, produced by site-directed mutagenesis. It will also be studied in vivo, by correlating phosphorylation with protein synthesis rates and by using transient expression vectors containing mutagenized forms of eIF-4E cDNA. Finally, the structure of the various forms of eIF-4E mRNA will be examined, and the gene for this protein will be cloned and partially sequenced. R0GM33804 Selected functional properties that are characteristic of cytochrome c will be investigated and the possible endowment of this protein with new functional properties will be explored through the construction of a series of specifically designed mutants as follows: (1) The putative crystallographic identification of a substrate binding site on the surface of the Ser-82 variant will be evaluated by examination of the effect of relevant small molecules on the oxidation-reduction properties of the protein. (2) The role of the axial ligands in determining cytochrome function will be studied by analysis of Met-80 mutants. (3) The mechanism by which mutations at positions 38 and 82 alter the alkaline transition will be studied by pH-jump experiments, EPR spectroscopy, and electrostatics calculations. (4) The Takano Dickerson model for cytochrome c redox interconversion will be evaluated by study of recently constructed Thr-78 mutants. This residue is critical to the Takano-Dickerson model as it hydrogen- bonds to a crucial, internally-bound water molecule that is pivotal to their proposal. (5) The role of heme propionate-7 in regulating the reduction potential of the protein will be studied by consideration of a Tyr-48 mutant. Possible analysis of Tyr-48/Arg- 38 double mutants will be considered in this regard as well. (6) Further analysis of the multiple roles of Phe-82 will be analyzed by evaluation of several new mutants constructed at this position. (7) The origin of species differences between cytochromes will be considered through construction and characterization of loop insertion/deletion mutants which convert yeast iso-l cytochrome c into forms closer in size to two prokaryotic cytochromes. (8) The effects of selected mutations on the kinetics of electron transfer to physiological redox partner proteins will be studied. (9) A battery of spectroscopic techniques (NMR, CD/MCD, and time-resolved fluorescence spectroscopy) will be applied to selected mutants as dictated by their observed properties.