The Structural Dynamics of Translation Initiation

翻译起始的结构动力学

基本信息

批准号：
10457282
负责人：
Ruben L Gonzalez
金额：
$ 33.62万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10457282
关键词：
Acceleration Address Anti-Bacterial Agents Antibiotics Antineoplastic Agents Attention Automobile Driving Base Pairing Binding Biochemical Clinical Collaborations Collection Complement Complex Cryoelectron Microscopy DNA Development Electron Microscopy Ensure Escherichia coli Fluorescence Microscopy Funding GTP Binding Gene Expression Goals Guanosine Homologous Gene Human Image In Vitro Initiator Codon Initiator tRNA Israel Kinetics Label Laboratories Letters Link Malignant Neoplasms Mediating Messenger RNA Modeling Molecular Molecular Conformation Molecular Medicine Organism Pathway interactions Peptide Initiation Factors Pharmaceutical Preparations Play Positioning Attribute Process Protein Biosynthesis Proteins Reagent Regulation Reporting Resolution Ribosomes Role Saccharomyces cerevisiae Series Signal Transduction Site Structural Models Structure System Techniques Time Transfer RNA Translating Translation Initiation Translations Variant Viral Cancer Virus Diseases base cryogenics experimental study fluorescence imaging fluorophore human disease next generation novel strategies pathogen prevent public health relevance recruit single molecule single-molecule FRET small molecule transmission process tumorigenesis

项目摘要

PROJECT SUMMARY The process through which the two-subunit ribosome assembles at the start codon of an mRNA to initiate protein synthesis is one of the most fundamental and highly regulated steps of gene expression. As such, initiation serves as the target of numerous small-molecule antibiotics and cellular pathogens. Moreover, deregulation of initiation is causally linked to viral infections and tumorigenesis in humans. Given all of this, studies of the molecular mechanism of initiation hold great promise for the identification and characterization of mechanistic steps that can serve as targets for the development of next-generation antibiotics and other small- molecule, anti-viral, and anti-cancer drugs that act by modulating translation initiation. Despite their promise for molecular medicine, mechanistic studies of initiation remain incredibly challenging. This is primarily because initiation is an extraordinarily dynamic, multi-step process that proceeds through a large number of short-lived intermediate states that are very difficult to observe and characterize using conventional approaches. During initiation, a set of essential initiation factors (IFs) transiently interact with both ribosomal subunits and a specialized initiator tRNA in order to guide their assembly at the start codon of the mRNA to be translated. Although evidence suggests that the IFs, ribosomal subunits, and tRNA undergo functionally important structural rearrangements during this process, very few of these rearrangements have been directly observed and/or characterized, and for those that have, it has been at very low resolution. The long-term goals of this project are to use powerful combinations of reagents and techniques that are uniquely available in our and our collaborators’ laboratories to overcome the challenges associated with mechanistic studies of initiation. Specifically, we will use state-of-the-art single-molecule fluorescence microscopy and cryogenic electron microscopy (cryo-EM), including a pioneering, time-resolved cryo-EM approach developed by our collaborator, Dr. Joachim Frank, to directly observe and characterize the dynamics of initiation. These studies will be enabled by a new approach that we have developed for introducing fluorophores into ribosomes at positions that are highly desirable, but that have thus far remained out of reach. In Aim 1, we will investigate the mechanism through which bacterial IF2 transiently binds to a ribosomal initiation complex (IC) based on the small, 30S, ribosomal subunit (30S IC); determines whether the 30S IC is carrying an accurately selected initiator tRNA that is properly base-paired to a correctly selected start codon; and, if so, recruits and facilitates joining of the large, 50S, ribosomal subunit to the 30S IC. In Aim 2, we will investigate the mechanism through which bacterial IF3 and IF1 ensure the accuracy with which the start codon is selected during initiation. In Aim 3, we will extend our studies to investigate the mechanism of eukaryotic translation initiation, focusing our attention on eukaryotic-specific aspects of the mechanism through which the eukaryotic homolog of IF2, eukaryotic IF5B (eIF5B), regulates subunit joining during eukaryotic initiation.

项目概要两个亚基核糖体在 mRNA 的起始密码子处组装成的过程启动蛋白质合成是基因表达最基本且高度调控的步骤之一。因此，起始是许多小分子抗生素和细胞病原体的靶标。鉴于所有这些，起始的放松管制与人类的病毒感染和肿瘤发生存在因果关系。引发分子机制的研究对于鉴定和表征具有广阔的前景机械步骤可以作为开发下一代抗生素和其他小型药物的目标通过调节翻译起始起作用的分子、抗病毒和抗癌药物。尽管它们对分子医学有希望，但起始机制研究仍然令人难以置信这主要是因为启动是一个异常动态、多步骤的过程。通过大量难以观察和表征的短暂中间状态在启动过程中，一组重要的启动因素（IF）会短暂地相互作用。具有核糖体亚基和专门的起始子 tRNA，以指导它们在起始密码子处的组装尽管有证据表明 IF、核糖体亚基和 tRNA 会发生翻译。在此过程中功能上重要的结构重排，这些重排中很少有被直接观察和/或表征，对于那些已经观察到和/或表征的人来说，它的分辨率非常低。该项目的长期目标是使用强大的试剂和技术组合，我们和我们合作者的实验室有独特的解决方案，可以克服与具体来说，我们将使用最先进的单分子荧光研究。显微镜和低温电子显微镜 (cryo-EM)，包括开创性的时间分辨冷冻电镜我们的合作者 Joachim Frank 博士开发的方法，用于直接观察和表征动态这些研究将通过我们为引入而开发的新方法来实现。荧光团进入核糖体的位置非常理想，但迄今为止仍然遥不可及。在目标 1 中，我们将研究细菌 IF2 短暂结合核糖体的机制基于小 30S 核糖体亚基 (30S IC) 的起始复合物 (IC) 确定 30S IC 是否为携带准确选择的起始tRNA，该tRNA与正确选择的起始密码子正确碱基配对；如果是这样，我们将招募并促进大型 50S 核糖体亚基与 30S IC 的连接。研究细菌 IF3 和 IF1 确保起始密码子准确性的机制在目标3中，我们将扩展我们的研究以调查真核生物的机制。翻译起始，将我们的注意力集中在真核生物特异性的机制上，通过该机制 IF2 的真核同源物真核 IF5B (eIF5B) 在真核起始过程中调节亚基连接。

项目成果

期刊论文数量（49）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation.

延伸因子 G 和核糖体 L1 柄之间的变构协作指导翻译过程中的 tRNA 运动。

DOI：
发表时间：
2009-09-15
期刊：
影响因子：
11.1
作者：
Fei, Jingyi;Bronson, Jonathan E;Hofman, Jake M;Srinivas, Rathi L;Wiggins, Chris H;Gonzalez Jr, Ruben L
通讯作者：
Gonzalez Jr, Ruben L

A microfluidic approach for investigating the temperature dependence of biomolecular activity with single-molecule resolution.

一种以单分子分辨率研究生物分子活性的温度依赖性的微流体方法。

DOI：
发表时间：
2011-01-21
期刊：
影响因子：
6.1
作者：
Wang, Bin;Ho, Joseph;Fei, Jingyi;Gonzalez Jr, Ruben L;Lin, Qiao
通讯作者：
Lin, Qiao

Ribosomal initiation complex-driven changes in the stability and dynamics of initiation factor 2 regulate the fidelity of translation initiation.

核糖体起始复合物驱动的起始因子 2 的稳定性和动态变化调节翻译起始的保真度。

DOI：
发表时间：
2015-05-08
期刊：
影响因子：
5.6
作者：
Wang, Jiangning;Caban, Kelvin;Gonzalez Jr, Ruben L
通讯作者：
Gonzalez Jr, Ruben L

Selective biomolecular nanoarrays for parallel single-molecule investigations.

用于并行单分子研究的选择性生物分子纳米阵列。

DOI：
10.1021/ja201031g
发表时间：
2011-05-25
期刊：
Journal of the American Chemical Society
影响因子：
15
作者：
Palma, Matteo;Abramson, Justin J.;Gorodetsky, Alon A.;Penzo, Erika;Gonzalez, Ruben L., Jr.;Sheetz, Michael P.;Nuckolls, Colin;Hone, James;Wind, Shalom J.
通讯作者：
Wind, Shalom J.

The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence.

警报酮 (p)ppGpp 在进入静止期间直接调节翻译起始。