Direct three-dimensional localization and positive identification of RNA helices within the ribosome by means of genetic tagging and cryo-electron microscopy.
Direct three-dimensional localization and positive identification of RNA helices within the ribosome by means of genetic tagging and cryo-electron microscopy.
复制标题
通过基因标记和冷冻电子显微镜对核糖体内的 RNA 螺旋进行直接三维定位和阳性鉴定。
DOI：
10.1016/s0969-2126(00)88347-1
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Frank,J
中科院分区：
文献类型：
--
作者：
Spahn,CM;Grassucci,RA;Penczek,P;Frank,J
背景：核糖体是执行遗传信息翻译的复杂的大分子机器。大肠杆菌70S核糖体的冷冻电子显微镜(CRYO-EM)图谱的分辨率接近10？，30S和50S亚基的X射线图谱现已在5？处可用。这些地图显示了核糖体内部结构的许多细节，核糖体RNA螺旋清晰可见。然而，在缺乏进一步的生物学信息的情况下，即使在X射线图的较高分辨率下，许多rRNA螺旋也只能暂时放置。在这里，我们证明了基因标签和冷冻-EM相结合可以高精度地定位和定位双链RNA螺旋。结果：将tRNA序列插入到TERE中。Coli23S核糖体RNA基因位于真核细胞核糖体的序列扩张点之一，在冷冻-EM图谱上可见其为外围的“脚”结构。通过追踪其受体-茎末端，可以精确地确定螺旋63在50S亚基的IV区和螺旋98在VI区的位置。结论：我们的研究首次证明了不对称大分子复合体的三维冷冻-EM图谱的特征可以从二级结构和一级结构的角度进行解释。使用已识别的螺旋作为起点，就有可能以分子的形式对核糖体的更大部分进行建模和解释。我们的结果可能对解释和改进当前的X射线图也很有用。
Background:Ribosomes are complex macromolecular machines that perform the translation of the genetic message. Cryo-electron microscopic (cryo-EM) maps of theEscherichia coli70S ribosome are approaching a resolution of 10 Å and X-ray maps of the 30S and 50S subunits are now available at 5 Å. These maps show a lot of details about the inner architecture of the ribosome and ribosomal RNA helices are clearly visible. However, in the absence of further biological information, even at the higher resolution of the X-ray maps many rRNA helices can be placed only tentatively. Here we show that genetic tagging in combination with cryo-EM can place and orient double-stranded RNA helices with high accuracy.Results:A tRNA sequence inserted into theE. coli23S ribosomal RNA gene, at one of the points of sequence expansion in eukaryotic ribosomes, is visible in the cryo-EM map as a peripheral ‘foot' structure. By tracing its acceptor-stem end, the location of helix 63 in domain IV and helix 98 in domain VI of the 50S subunit could be precisely determined.Conclusions:Our study demonstrates for the first time that features of a three-dimensional cryo-EM map of an asymmetric macromolecular complex can be interpreted in terms of secondary and primary structure. Using the identified helices as a starting point, it is possible to model and interpret, in molecular terms, a larger portion of the ribosome. Our results might be also useful in interpreting and refining the current X-ray maps.