STUDIES OF TRANSLATION IN E COLI IN THE PHASES OF INITIATION, DECODING,

大肠杆菌翻译起始阶段、解码阶段、

• 批准号: 7954564
• 负责人: JOACHIM FRANK
• 金额: $ 3.35万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954564
• 关键词: Acetylation; Amino Acyl Transfer RNA; Binding; Biological; Biopolymers; Cells; Characteristics; Classification; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Data Collection; Data Set; Docking; EEF1A1 gene; EEF2 gene; Electron Microscopy; Electrons; Elongation Factor; Enzymatic Biochemistry; Escherichia coli; Event; Funding; Goals; Grant; Guanosine Triphosphate; Head; Hydrolysis; Image; Imagery; Institution; Introns; Ligand Binding; Macromolecular Complexes; Mediating; Methods; Microscopic; Modeling; Molecular; Molecular Conformation; Motion; Movement; Nature; Paper; Pathway interactions; Peptide Elongation Factor G; Phase; Positioning Attribute; Preparation; Process; Proteins; Protocols documentation; RNA; Reaction; Research; Research Infrastructure; Research Personnel; Resolution; Resources; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Rotation; Science; Site; Source; Specimen; Structure; Sweden; Techniques; Time; Transfer RNA; Translation Initiation; Translations; United States National Institutes of Health; Work; X-Ray Crystallography; Yeasts; abstracting; base; computerized data processing; image processing; improved; insight; macromolecule; men' s group; molecular mechanics; particle; protein B; reconstruction; ribosomal protein S1; ribosome releasing factor; scaffold; simulation; termination factor; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Cryo-EM single-particle reconstruction has evolved to become the most powerful approach to study ligand binding and conformational changes accompanying translation. A recent review (Frank, 2003) gives an overview over the many results that have accumulated since 1996, when tRNA binding to the ribosome was first visualized (Agrawal et al., 1996). EF-G mediated translocation was the first focus of the PI's research (Agrawal et al., 1998; 1999; Frank and Agrawal, 2000), yielding three significant findings (i) EF-G binds to the ribosome in a position that is very similar to the binding position of the aminoacyl-tRNA-EF-Tu-GTP complex; (ii) EF-G undergoes a conformational change, characterized by a rotation of domains III-V; and (iii) the ribosome undergoes a "ratchet" motion, characterized by a rotation of the small subunit against the large subunit by as much as 10 degrees. Since the year 2000, we have collaborated with the group of Mans Ehrenberg in Uppsala, Sweden, enabling us to look at highly purified, kinetically characterized translational complexes in all different phases of translation. This collaboration has resulted in a number of major discoveries that have enriched our understanding of the molecular events. A partial list is given here briefly in telegraph style: (i) Decoding and tRNA accommodation are accompanied by a large change in tRNA conformation (Valle et al., 2002; 2003a); (ii) The binding of a variety of factors to the ribosome leads to a characteristic conformational change of the stalk base (Rawat et al., 2003; Valle et al., 2003a); (iii) Binding of the tRNA D-loop to the L11-rRNA complex, observed by Valle and coworkers (2003a) is apparently facilitated by the flipping-out of a base of the rRNA (Li et al., submitted); (iv) The ratchet motion is a universal mechanism, observed with the binding of EF-G (Frank and Agrawal, 2000), EF2 to the 80S ribosome from yeast (Spahn et al., 2004), RF3 (U. Rawat et al., in preparation), and RRF (N. Gao et al., in preparation). (v) The ratchet motion involves an "elastic" deformation of the entire RNA matrix, and large movements and conformational changes of a number of ribosomal proteins (Gao et al., 2003); (vi) The ribosome can change from an "unlocked" state to a "locked" state, and this change is controlled by the state of acetylation of the P-site tRNA (Valle et al., 2003b); (vii) The L1 stalk is highly mobile, and its movement is anticorrelated with the movement of the small subunit head, such that the intersubunit space is opened and closed (Valle et al., 2003b). [While the movement involves a single hinge in the case of bacterial ribosomes (Valle et al., 2003), there are two hinges in the case of the eukaryotic ribosome (Spahn et al., 2004)]; (viii) When bound to the ribosome, release factors RF2 (Rawat et al., 2003) and RF1 (Rawat et al., in preparation) assume a conformation strongly different from that observed for RF2 by X-ray crystallography; (ix) RF3 has a conformation and binding position strikingly similar to those of EF-G (Rawat et al., in preparation). Using existing tools of cryo-EM single-particle reconstruction, and tools being developed in TRD2 and TRD3, we wish to pursue these studies with improved resolution, and follow the reaction pathways at increasing levels of detail. The ultimate goal is the description and understanding of molecular mechanisms underlying these processes. The experimental protocols for specimen preparation, electron microscopy, data processing, and interpretation have been described (Frank et al., 2000; Frank, 2002; Frank, 1996). Various time-resolved techniques outlined in TRD2 will be tried to capture additional states of the ribosome in the processes we have characterized. Resolution will be increased, by the use of automated data collection, improved image processing methods, and classification (TRD3), to improve the accuracy of docking and modeling of molecular interactions. Real-space refinement will be applied to determine the underlying molecular events, similar as has been done in Gao et al. (2003). Selected components of the ribosome will be studied by molecular mechanics simulations correlated with cryo-EM observations. References: 1. R.K. Agrawal, P. Penczek, R.A. Grassucci, Y. Li, A. Leith, K.H. Nierhaus, and J. Frank (1996) Direct visualization of A-, P-, and E-site transfer RNAs in the Escherichia coli ribosome. Science 271:1000-1002. 2. R.K. Agrawal, P. Penczek, R.A. Grassucci, and J. Frank (1998). Visualization of elongation factor G on the Escherichia coli ribosome: The mechanism of translocation. Proc. Natl. Acad. Sci. (USA) 95:6134-6138. 3. R.K. Agrawal, A.B. Heagle, P. Penczek, R.A. Grassucci, and J. Frank (1999) EF-G dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome. Nat. Struct. Biol. 6: 643-647. 4. J. Frank (1996) Three-dimensional Electron Microscopy of Macromolecular Complexes. Academic Press, San Diego. 5. J. Frank and R.K. Agrawal (2000) A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature, 406:318-322. 6. J. Frank, P. Penczek, R.K. Agrawal, R.A. Grassucci, and A.B. Heagle (2000) Three-dimensional cryoelectron microscopy of ribosomes. In Methods of Enzymology. Edited by D.W. Celander and J.N. Abelson, Academic Press, San Diego, CA. Chpt. 18, 276-291. 7. J. Frank (2003) Single-particle imaging of macromolecules by cryo-electron microscopy. Ann. Rev. Biophys. Biomol. Struct. 31:303-319. 8. J. Frank. (2003) Electron microscopy of functional ribosome complexes. Biopolymers 68: 223-233. 9. H. Gao, J. Sengupta, M. Valle, A. Korostelev, N. Eswar, S.M. Stagg, P. Van Roey, R.K. Agrawal, S.C. Harvey, A. Sali, M.S. Chapman, and J. Frank (2003) Study of the structural dynamics of the E. coli 70S ribosome using real space refinement. Cell 113:789-801. 10. H. Gao, M. Valle, M. Ehrenberg, and J. Frank (2004) Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset. J. Struct. Biol., in press. 11. U.B.S. Rawat, A.V. Zavialov, J. Sengupta, M. Valle, R.A. Grassucci, J. Linde, B. Vestergaard, M. Ehrenberg, and J. Frank. (2003) A cryo-electron microscopic study of ribosome-bound termination factor RF2, Nature 421:87-90. 12. C.M.T. Spahn, M.G. Gomez-Lorenzo, R.A. Grassucci, R. J¿rgensen, G.R. Andersen, R. Beckmann, P.A. Penczek, J.P.G. Ballesta, and J. Frank (2004) Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J., 23:1008-1019. 13. M. Valle, J. Sengupta, N.K. Swami, R.A. Grassucci, N. Burkhardt, K.H. Nierhaus, R.K. Agrawal, and J. Frank (2002) Cryo-EM reveals an active role for the aminoacyl-tRNA in the accomodation process. EMBL J., 21:3557-3567. 14. M. Valle, A. Zavialov, W. Li, S.M. Stagg, J. Sengupta, R.C. Nielsen, P. Nissen, S.C. Harvey, M. Ehrenberg, and J. Frank (2003a) Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-EM. Nature Struct. Biol., 10: 899-906. 15. M. Valle, A. Zavialov, J. Sengupta, U. Rawat, M. Ehrenberg, and J. Frank (2003b) Locking and unlocking of ribosomal motions. Cell, 114:123-134. The following papers, although not directly related to the RVBC, represent collaborations that benefited from the infrastructure provided by the RVBC: + Allen GS, Frank J (2007) Structural insights on the translation initiation complex: ghosts of a universal initiation complex. Molec Microbiol 63: 941-950. + Gillet R, Kaur S, Li W, Hallier M, Felden B, Frank J (2007) Scaffolding as an organizing principle in trans-translation: The roles of small protein B and ribosomal protein S1. J. Biol. Chem. 282, 6356-6363. + Mitra K, Frank J, Driessen A (2006) Co- and post-translational translocation through the protein-conducting channel: Analogous mechanisms at work? Nature Struct Mol Biol 13: 957-964. + Slagter-Jager JG, Allen GS, Smith D, Hahn IA, Frank J, Belfort M (2006) Visualization of a group II introns in the 23S rRNA of a stable ribosome. PNAS 103, 9838-9843. + Taylor DJ, Nilsson J, Merrill AR, Andersen GR, Nissen P, Frank J (2007) Structures of modified eEF2 + 80S ribosome complexes reveal the role of GTP hydrolysis in translocation. EMBO J. 26, 2421-2431.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 抽象的： 冷冻电镜单粒子重建已发展成为研究配体结合和伴随翻译的构象变化的最强大方法。最近的一篇评论（Frank，2003）概述了自 1996 年 tRNA 结合以来积累的许多结果。核糖体首次被可视化（Agrawal et al., 1996）。EF-G 介导的易位是 PI 研究的第一个焦点（Agrawal et al., 1996）。 1998；1999；Frank 和 Agrawal，2000），得出三个重要发现 (i) EF-G 与核糖体的结合位置与氨酰基-tRNA-EF-Tu-GTP 复合物的结合位置非常相似； ) EF-G 经历构象变化，其特征是结构域 III-V 的旋转；以及 (iii) 核糖体经历“棘轮”运动，其特征是结构域 III-V 的旋转；自 2000 年以来，我们与瑞典乌普萨拉的 Mans Ehrenberg 团队合作，使小亚基与大亚基相差多达 10 度，使我们能够在所有不同阶段观察高度纯化的、具有动力学特征的翻译复合物。这次合作产生了许多重大发现，丰富了我们对分子事件的理解，这里以电报的形式简要列出了部分内容： (i) 解码和 tRNA 调节伴随着 tRNA 构象的巨大变化（Valle 等，2002；2003a）； (ii) 多种因子与核糖体的结合导致茎基部的特征性构象变化（Rawat 等人，2003；Valle 等人，2003a）； (iii) Valle 和同事 (2003a) 观察到，rRNA 碱基的翻转显然促进了 tRNA D 环与 L11-rRNA 复合物的结合（Li 等人提交）； (iv) 棘轮运动是一种通用机制，通过 EF-G（Frank 和 Agrawal，2000）、EF2 与酵母 80S 核糖体（Spahn 等人，2004）、RF3（U. Rawat 等人）的结合观察到。 .，正在准备中）和 RRF（N.Gao 等人，正在准备中）。 (v) 棘轮运动涉及整个RNA基质的“弹性”变形，以及许多核糖体蛋白的大运动和构象变化（Gao等，2003）； (vi) 核糖体可以从“解锁”状态变为“锁定”状态，这种变化是由 P 位 tRNA 的乙酰化状态控制的（Valle et al., 2003b）； (vii) L1 柄具有高度移动性，其运动与小亚基头的运动反相关，从而使亚基间空间打开和关闭（Valle et al., 2003b）[虽然运动涉及单个铰链]。就细菌核糖体而言（Valle et al., 2003），就真核核糖体而言有两个铰链（Spahn et al., 2003）。 2004)]; (viii) 当与核糖体结合时，强烈释放因子 RF2（Rawat 等人，2003）和 RF1（Rawat 等人，准备中）呈现与通过 X 射线晶体学观察到的 RF2 不同的构象； (ix) RF3 的构象和结合​​位置与 EF-G 惊人地相似（Rawat 等人，正在准备中）。 使用现有的冷冻电镜单粒子重建工具以及 TRD2 和 TRD3 中开发的工具，我们希望以更高的分辨率进行这些研究，并以越来越详细的水平跟踪反应途径，最终目标是描述和理解。这些过程背后的分子机制。 已经描述了样品制备、电子显微镜、数据处理和解释的实验方案（Frank et al., 2000; Frank, 2002; Frank, 1996）。TRD2 中概述的各种时间分辨技术将尝试捕获其他状态。通过使用自动数据收集、改进的图像处理方法和分类 (TRD3)，我们将提高核糖体在我们表征的过程中的分辨率，以提高对接和建模的准确性。真实空间细化将用于确定潜在的分子事件，类似于Gao等人（2003）将通过与冷冻电镜观察相关的分子力学模拟来研究核糖体的选定成分。 。 参考： 1. R.K. Agrawal、P. Penczek、R.A. Grassucci、Y. Li、A. Leith、K.H. Nierhaus 和 J. Frank (1996) 大肠杆菌核糖体中 A、P 和 E 位点转移 RNA 的直接可视化科学 271：1000-1002。 2. R.K. Agrawal、P. Penczek、R.A. Grassucci 和 J. Frank (1998) 大肠杆菌核糖体上的延伸因子 G 的可视化：《国家科学》杂志。 -6138。 3. R.K. A.B. Heagle、P. Penczek、R.A. Grassucci 和 J. Frank (1999) EF-G 依赖性 GTP 水解引起 70S 核糖体构象变化。 647. 4. J. Frank (1996) 大分子复合物的三维电子显微镜，圣地亚哥学术出版社。 5. J. Frank 和 R.K. Agrawal (2000) 易位期间核糖体亚基间的棘轮重组，《自然》，406：318-322。 6. J. Frank、P. Penczek、R.A. Grassucci 和 A.B. Heagle（《核糖体三维冷冻电子显微镜》），D.W. Celander 和 J.N Abelson 编辑。第 18 章，276-291。 7. J. Frank (2003) 通过冷冻电子显微镜进行的单粒子成像。Ann. Biophys. 31:303-319。 8. J. Frank (2003) 功能性核糖体复合物的电子显微镜 68：223-233。 9. H. 高、J. Sengupta、M. Valle、A. Korostelev、N. Eswar、S.M. Stagg、P. Van Roey、R.K. Agrawal、S.C. Harvey、A. Sali、M.S. Chapman 和 J. Frank使用真实空间细化研究大肠杆菌 70S 核糖体的结构动力学。 113：789-801。 10. H. Gau、M. Valle、M. Ehrenberg 和 J. Frank (2004) 通过异质冷冻电镜数据集分类探索 EF-G 与核糖体相互作用的动力学，出版中。 。 11. U.B.S. Rawat、A.V. Zavialov、J. Sengupta、M. Valle、R.A. Linde、B. Vestergaard、M. Ehrenberg 和 J. Frank（核糖体结合终止的冷冻电子显微镜研究）因子 RF2，《自然》421：87-90。 12. C.M.T. 斯潘，M.G. 戈麦斯-洛伦佐，R.A. 格拉苏奇rgensen、G.R. Andersen、P.A. Penczek、J.P.G. Ballesta 和 J. Frank (2004) 延伸因子 eEF2 和真核 80S 核糖体的结构域运动促进 tRNA 易位。 13. M. Valle、J. Sengupta、N.K. Swami、R.A. Grassucci、N. Burkhardt、K.H. Nierhaus、R.K. Agrawal 和 J. Frank (2002) 冷冻电镜揭示了氨酰基-tRNA 在调节过程中的积极作用。 EMBL J.，21：3557-3567。 14. M. Valle、A. Zavialov、W. Li、S.M. Stagg、J. Sengupta、R.C. Nielsen、P. Nissen、S.C. Harvey、M. Ehrenberg 和 J. Frank (2003a) 氨酰基-tRNA 并入核糖体正如冷冻电镜所见。《自然结构》，10：899-906。 15. M. Valle、A. Zavialov、J. Sengupta、U. Rawat、M. Ehrenberg 和 J. Frank (2003b) 核糖体运动的锁定和解锁，114：123-134。 以下论文虽然与 RVBC 没有直接关系，但代表了受益于 RVBC 提供的基础设施的合作： + Allen GS、Frank J (2007) 对翻译起始复合物的结构见解：通用起始复合物的幽灵 63：941-950。 + Gillet R、Kaur S、Li W、Hallier M、Felden B、Frank J (2007) 支架作为反式翻译的组织原理：小蛋白 B 和核糖体蛋白 S1 的作用。《生物杂志》282。 6356-6363。 + Mitra K、Frank J、Driessen A (2006) 通过蛋白质传导通道的共翻译和翻译后易位：工作中的类似机制？ Nature Struct Mol Biol 13：957-964。 + Slagter-Jager JG、Allen GS、Smith D、Hahn IA、Frank J、Belfort M (2006) 稳定核糖体 23S rRNA 中 II 组内含子的可视化，103, 9838-9843。 + Taylor DJ、Nilsson J、Merrill AR、Andersen GR、Nissen P、Frank J (2007) 修饰的 eEF2 + 80S 核糖体复合物的结构揭示了 GTP 水解在易位中的作用。 26, 2421-2431。