X-RAY STRUCTURES OF EUKARYOTIC TRANSLATION FACTORS

真核翻译因子的 X 射线结构

• 批准号: 6363350
• 负责人: JEFFREY B BONANNO
• 金额: $ 39.54万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363350
• 关键词: ADP ribosylation; Picornaviridae; RNA; X ray crystallography; adenosine diphosphate; bioimaging /biomedical imaging; eukaryote; guanine nucleotide binding protein; guanosine diphosphate; helicase; messenger RNA; methionine; polyadenylate; ribosomes; transfer RNA; translation factor; virus RNA

The long-term goal of the proposed research is a detailed structural and mechanistic understanding of the enormously complex macromolecular machine that controls translation initiation in eukaryotes. The proposed work builds on our structural and functional studies of mRNA selection by the cap-binding protein, eukaryotic initiation factor 4E or eIF4E. X-ray crystallography will be combined with chemical methods to study the structures and mechanisms of action of an important subset of the remaining translation initiation factors [eIF4G, eIF4A, eIF2, IF2, eIF3, eIF1A, eIF2B, eIF5, the poly (A)-binding protein (PABP), and a PABP-interacting protein]. These translation factors participate in the following biochemical steps: (a) resolving secondary structural features in the 5' untranslated region of the mRNA, (b) delivering the methionyl initiator tRNA to the 40S subunit, (d) recycling the G protein that loads the methionyl initiator tRNA onto the 40S subunit, (d) recruitment of the 40S subunit, the methionyl initiator tRNA and various accessory factors to the 5' untranslated region of cellular mRNAs bearing 5' 7-methyl-G caps, (e) recruitment of the same components to the 5' untranslated region of an uncapped viral RNA, (f) 60S ribosomal subunit joining, and (g) synergy of transcription initiation via mRNA circularization by the PABP. The specific aims of the research are as follows: Specific Aim 1. Determine X-ray structures of the RNA helicase eIF4A, and its complexes with ADP, a non-hydrolyzable ATP analog, and single-stranded RNA. Specific Aim 2. Determine X-ray structures of eIF2, and its complexes with GDP, a non-hydrolyzable GTP analog, and methionyl initiator tRNA. Specific Aim 3. Determine the X-ray structure of eukaryotic IF2. Specific Aim 4. Determine X-ray structures of a C-terminal fragment of eIF4G, and its complex with a picornavirus internal ribosome entry site. Specific Aim 5. Determine X-ray structures of eIF5 and eIF2Bepsilon, and their complexes with eIF2beta. Specific Aim 6. Determine X-ray structures of PABP recognizing poly (A) RNA, and various binary and ternary complexes with a PABP-interacting protein and eIF4G.

拟议研究的长期目标是对控制真核生物翻译起始的极其复杂的大分子机器进行详细的结构和机制理解。 拟议的工作建立在我们通过帽结合蛋白、真核起始因子 4E 或 eIF4E 对 mRNA 选择的结构和功能研究的基础上。 X 射线晶体学将与化学方法相结合，研究剩余翻译起始因子的一个重要子集的结构和作用机制 [eIF4G、eIF4A、eIF2、IF2、eIF3、eIF1A、eIF2B、eIF5、聚 (A) -结合蛋白(PABP)和PABP相互作用蛋白]。 这些翻译因子参与以下生化步骤：(a) 解析 mRNA 5' 非翻译区的二级结构特征，(b) 将甲硫氨酰起始子 tRNA 递送至 40S 亚基，(d) 回收加载 40S 亚基的 G 蛋白。甲硫氨酰起始子 tRNA 附着到 40S 亚基上，(d) 将 40S 亚基、甲硫氨酰起始子 tRNA 和各种辅助因子募集到 40S 亚基上。带有 5' 7-甲基-G 帽的细胞 mRNA 的 5' 非翻译区，(e) 将相同成分募集到无帽病毒 RNA 的 5' 非翻译区，(f) 60S 核糖体亚基连接，以及 (g) 协同作用PABP 通过 mRNA 环化转录起始。本研究的具体目的如下： 具体目的1.确定RNA解旋酶eIF4A及其与不可水解ATP类似物ADP和单链RNA的复合物的X射线结构。 具体目标 2. 确定 eIF2 及其与 GDP、不可水解 GTP 类似物和甲硫氨酰引发剂 tRNA 的复合物的 X 射线结构。 具体目标 3. 确定真核生物 IF2 的 X 射线结构。 具体目标 4. 确定 eIF4G 的 C 末端片段及其与小核糖核酸病毒内部核糖体进入位点的复合物的 X 射线结构。具体目标 5. 确定 eIF5 和 eIF2Bepsilon 及其与 eIF2beta 的复合物的 X 射线结构。 具体目标 6. 确定识别 Poly (A) RNA 的 PABP 的 X 射线结构，以及具有 PABP 相互作用蛋白和 eIF4G 的各种二元和三元复合物。