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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 抽象的： 冷冻EM单粒子重建已发展为研究配体结合和构象变化涉及翻译的最强大方法。最近的评论（Frank，2003）概述了自1996年以来积累的许多结果，当时TRNA与核糖体结合，首先被可视化（Agrawal等，1996）。 EF-G介导的翻译是PI研究的第一个焦点（Agrawal等，1998; 1999; 1999; Frank and Agrawal，2000），得出了三个重要发现（i）EF-G与核糖体在与氨基化位置非常相似的位置的核糖体结合； （ii）EF-G经历构象变化，其特征是域III-V的旋转； （iii）核糖体经历“棘轮”运动，其特征是小亚基与大亚基的旋转高达10度。自2000年以来，我们与瑞典Uppsala的Ehrenberg群体合作，使我们能够在所有不同的翻译阶段中查看高度纯化的，动力学的翻译成络合物。这种合作导致了许多重大发现，这些发现丰富了我们对分子事件的理解。这里简要列出了部分列表，以电报风格给出： （i）解码和tRNA的适应性伴随着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碱基的特征会议变化（Rawat等，2003； Valle等，2003a）； （iii）valle and Crocores（2003a）观察到的tRNA D环与L11-RRNA复合物的结合显然是由RRNA底座的翻转（Li等人，提交）的； （iv）棘轮运动是一种通用机制，观察到EF-G（Frank and Agrawal，2000），EF2与酵母中80年代核糖体的结合（Spahn等，2004），RF3（U. Rawat等人，在制备中）和RRF（N. Gao等人的准备）。 （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动作和构象变化（Gao等，2003）； （vi）核糖体可以从“解锁”状态变为“锁定”状态，并且这种变化受P点tRNA的乙酰化状态控制（Valle等，2003b）； （vii）L1茎高度流动，其运动与小亚基头的运动是反相关的，因此打开并封闭了亚基间空间（Valle等，2003b）。 [虽然在细菌核糖体的情况下，该运动涉及一个铰链（Valle等，2003），但在真核核糖体的情况下，有两个铰链（Spahn等，2004）]； （viii）当与核糖体结合时，释放因子RF2（Rawat等，2003）和RF1（Rawat等人，在制备中）假定的会议与X射线晶体学观察到的RF2截然不同。 （IX）RF3具有与EF-G的构象和结合​​位置的相似之处（Rawat等人，在制备中）。 使用Cryo-EM单粒子重建的现有工具，以及在TRD2和TRD3中开发的工具，我们希望以改进的分辨率进行这些研究，并以越来越多的细节水平遵循反应途径。最终目标是对这些过程背后的分子机制的描述和理解。 已经描述了用于标本制备，电子显微镜，数据处理和解释的实验方案（Frank等，2000； Frank，2002； Frank，1996）。 TRD2中概述的各种时间分辨技术将尝试在我们表征的过程中捕获核糖体的其他状态。通过使用自动数据收集，改进的图像处理方法和分类（TRD3）来提高分子相互作用和建模的准确性，从而提高分辨率。实际空间的细化将用于确定基础分子事件，与Gao等人相似。 （2003）。核糖体的选定成分将通过与冷冻EM观测值相关的分子力学模拟研究。 参考： 1。R.K。 Agrawal，P。Penczek，R.A。 Grassucci，Y。Li，A。Leith，K.H。 Nierhaus和J. Frank（1996）在大肠杆菌核糖体中直接可视化A-，P-和E位置转移RNA。科学271：1000-1002。 2。R.K。 Agrawal，P。Penczek，R.A。 Grassucci和J. Frank（1998）。大肠杆菌核糖体上伸长因子g的可视化：易位的机理。 Proc。纳特。学院。科学。 （美国）95：6134-6138。 3。R.K。 A.B. Agrawal Heagle，P。Penczek，R.A。 Grassucci和J. Frank（1999）EF-G依赖性GTP水解诱导易位，伴随着70S核糖体的构象变化。纳特。结构。生物。 6：643-647。 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 and R.K. Agrawal（2000）在易位过程中核糖体的棘轮中的棘轮中的重组。自然，406：318-322。 6. J. Frank，P。Penczek，R.K。 Agrawal，R.A。 Grassucci和A.B. Heagle（2000）核糖体的三维冷冻电子显微镜。在酶学方法中。由D.W.编辑Celander和J.N.阿伯森，学术出版社，加利福尼亚州圣地亚哥。 chpt。 18，276-291。 7。J.Frank（2003）通过冷冻电子显微镜对大分子的单粒子成像。安。 Biophys牧师。生物元。结构。 31：303-319。 8。J.Frank。 （2003）功能性核糖体复合物的电子显微镜。生物聚合物68：223-233。 9. H. Gao，J。Sengupta，M。Valle，A。Korostelev，N。Eswar，S。M。Stagg，P。VanRoey，R。K。Agrawal，S。C。Harvey，A。Sali，A。Sali，M。S。Chapman，M。S。Chapman和J. Frank（2003）研究了E. Coli 70S Ribosome的结构动力学，使用真实的空间修补。牢房113：789-801。 10。H.Gao，M。Valle，M。Ehrenberg和J. Frank（2004）通过异质性冷冻数据集的分类探索的EF-G相互作用与核糖体相互作用的动力学。 J. struct。 Biol。，印刷中。 11. U.B.S.拉瓦特（A.V.） Zavialov，J。Sengupta，M。Valle，R.A。 Grassucci，J。Linde，B。Vestergaard，M。Ehrenberg和J. Frank。 （2003）核糖结合终止因子RF2的低温电子显微镜研究，自然421：87-90。 12.C.M.T. Spahn，M.G。 R.A. Gomez-Lorenzo Grassucci，R.J¿Rgensen，G.R。 Andersen，R。Beckmann，P.A。 Penczek，J.P.G。 Ballesta和J. Frank（2004）伸长因子EEF2和真核80年代核糖体的域运动促进tRNA易位。 Embo J.，23：1008-1019。 13。M.Valle，J。Sengupta，N.K。斯瓦米，R.A。 Grassucci，N。Burkhardt，K.H。 Nierhaus，R.K。 Agrawal和J. Frank（2002）Cryo-EM揭示了氨基酰基-TRNA在住宿过程中的积极作用。 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）将氨基酰基TRNA掺入核糖体中，如Cryo-Em所见。自然结构。 Biol。，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。牢房，114：123-134。 以下论文虽然与RVBC没有直接相关，但代表了从RVBC提供的基础架构中受益的合作： + Allen GS，Frank J（2007）关于翻译起始综合体的结构见解：普遍启动综合体的幽灵。 Molec Microbiol 63：941-950。 + Gillet R，Kaur S，Li W，Hallier M，Felden B，Frank J（2007）将脚手架作为跨翻译中的组织原理：小蛋白B和核糖体蛋白S1的作用。 J. Biol。化学282，6356-6363。 + Mitra K，Frank J，Driessen A（2006）通过蛋白质传导通道的转运和后翻译易位：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）在稳定的核糖体23S rRNA中可视化II组方法。 PNAS 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。 Embo J. 26，2421-2431。