REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITIATION

真核蛋白质合成起始的调控

• 批准号: 3270171
• 负责人: ROBERT E. RHOADS
• 金额: $ 22万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-04-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270171
• 关键词: affinity chromatography; affinity labeling; binding proteins; cell free system; chemical binding; chickens; complementary DNA; cytochrome c; estrogens; fallopian tubes; gel electrophoresis; genetic manipulation; genetic transcription; genetic translation; glycoproteins; guanosine triphosphate; high performance liquid chromatography; immunochemistry; ion exchange chromatography; laboratory rabbit; messenger RNA; molecular cloning; nucleic acid chemical synthesis; nucleic acid inhibitor; nucleotide analog; ovalbumin; phosphorylation; point mutation; polysomes; protein biosynthesis; protein kinase; protein reconstitution; protein sequence; protein structure function; reticulocytes; tissue /cell culture; transposon /insertion element

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.

真核生物中蛋白质合成的起始是 通过最复杂的一系列生化过程之一完成 反应已知。 了解这个过程对于 了解蛋白质合成速率是如何调节的， 许多病毒的细胞毒性作用，抗病毒药物的作用 蛋白质干扰素，以及基因的最终表达 信息。 该项目的长期目标是两个—— 折叠：了解各种蛋白质的生化作用（elF- 4组起始因子）参与信使RNA的进入 进入启动过程，以及负责的启动因素 用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，主动 eIF-4E 的位点将通过两种技术的组合来确定： 将合成一组新的光亲和标签并用于 标记 eIF-4E 的 m7GTP 结合位点。 二、改变形式 eIF-4E 将通过定点诱变在体外产生。 这 eIF-4A、-4B、-4E 和 -4F 在各种起始条件下的位置 复合物将被确定。 帽结构的相互作用， 在 m7GTP 的 mRNA 和光亲和衍生物中， eIF-4 群体因素将被调查。 最后，cDNA为 eIF-4F 的 p220 成分将被克隆并测序。 对于 第二个目标，eIF-4E磷酸化的效果将是 检查了。 这将通过使用体外研究 特定激酶和 eIF-4E 形式的无细胞合成 缺乏磷酸化位点，由定点产生 诱变。 它还将在体内进行研究，通过关联 磷酸化与蛋白质合成率和使用瞬态 含有诱变形式的 eIF-4E cDNA 的表达载体。 最后，各种形式的 eIF-4E mRNA 的结构将 进行检查，该蛋白质的基因将被克隆并 部分测序。 R0GM33804 选定的功能特性是 将研究细胞色素 c 并可能赋予 将探索这种具有新功能特性的蛋白质 通过建造一系列专门设计的 突变体如​​下： (1) 假定的晶体学 识别表面上的底物结合位点 Ser-82 变体将通过检查的效果进行评估 相关小分子对氧化还原性质的影响 蛋白质。 (2) 轴向配体在决定中的作用 将通过分析 Met-80 突变体来研究细胞色素功能。 (3) 38位和82位突变改变的机制 碱性转变将通过 pH 跳跃实验、EPR 研究 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 模型为 通过对最近构建的 Thr-78 突变体的研究进行评估。 这 残留物对于 Takano-Dickerson 模型至关重要，因为它含有氢 与关键的内部结合水分子结合，该水分子至关重要 他们的建议。 (五)heme propionate-7的调节作用 蛋白质的还原潜力将通过以下方式研究 考虑 Tyr-48 突变体。 Tyr-48/Arg- 的可能分析 在这方面也将考虑 38 个双突变体。 (6) 进一步分析Phe-82的多重作用 通过评估在该位置构建的几个新突变体。 (7) 细胞色素之间物种差异的起源将是 通过循环的构建和表征来考虑 转化酵母 iso-l 细胞色素 c 的插入/缺失突变体 形成尺寸更接近两种原核细胞色素的形式。 (8) 的 选定突变对电子转移动力学的影响 将研究生理氧化还原伙伴蛋白。 (9) 一个 一系列光谱技术（NMR、CD/MCD 和时间分辨 荧光光谱）将应用于选定的突变体 由它们观察到的特性决定。