来自大肠杆菌的翻译启动复合物的冷EM结构
Gregory S Allen1, Andrey Zavialov, Richard Gursky
1Howard Hughes Medical Institute, Health Research, Inc. at the Wadsworth Center, Albany, New York 12201, USA.
Cell
|June 7, 2005
概括
研究人员使用冷EM可视化了细菌翻译启动复合体. 他们观察了启动因子2 (IF2) 如何与核糖体和fMet-tRNA结合,揭示了对蛋白质合成至关重要的新型构造.
科学领域:
- 分子生物学分子生物学
- 结构生物学 结构生物学
- 微生物学 微生物学
背景情况:
- 细菌翻译启动是一个复杂的过程,涉及70S核糖体和多个启动因子.
- 了解这些成分的精确结构安排是解读蛋白质合成调节的关键.
研究的目的:
- 确定与GDPNP.停滞的细菌翻译启动复合体 (IC) 的高分辨率结构.
- 阐明IC中启动因子2 (IF2) 的作用和构成.
主要方法:
- 清洗大肠杆菌70S核糖体和翻译启动因子.
- 在实验室中使用GDPNP组装一个稳定的IC.
- 低温电子显微镜 (cryo-EM) 用于获得IC的3D重建.
主要成果:
- 一个冷-EM重建揭示了IF2*GDPNP位于70S核糖体的间子单元裂.
- IF2采用了一种新的形状,与之前确定的晶体结构不同.
- IF2的C终端域与fMet-tRNA ((fMet) 相互作用,将其单独定位在P位.
- IF2的GTP结合域与50S子单元的GTP酶相关中心相关,类似于延长因子.
结论:
- 这项研究为细菌翻译启动提供了前所未有的结构洞察力.
- IF2在稳定IC和通过独特的结构变化定位启动器tRNA方面发挥着关键作用.
- 其他因素,如IF1,IF3和核糖体蛋白的局部化也得到证据.
相关概念视频
Initiation of Translation
Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
Termination of Translation
The large ribosomal subunit has several important structures essential to translation. These include the peptidyl transferase center (PTC) - which is the site where the peptide bond is formed - and a large, internal, water-filled tube through which the nascent polypeptide moves. This latter structure is called the Peptide Exit Tunnel, and it begins at the PTC and spans the body of the large ribosomal subunit. During translation, as the nascent polypeptide chain is synthesized, it passes through...
Directing Proteins to the Rough Endoplasmic Reticulum
The organelle-specific signaling sequences direct proteins synthesized in the cytosol to their final destination like ER, mitochondria, peroxisomes, etc. Some of the proteins directed to ER are then trafficked via vesicles to other organelles within the cell or the extracellular environment through the Golgi complex. For example, the rough ER synthesizes soluble proteins for transportation to the lysosomes or secretion out of the cell. It can also synthesize transmembrane proteins that can...
Protein Translocation Machinery on the ER Membrane
The translocon complex situated on the ER membrane is the main gateway for the protein secretory pathway. It facilitates the transport of nascent peptides into the ER lumen and their insertion into the ER membrane.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.
Initiation of Translation
Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
Cryo-electron Microscopy
Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...


