The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography
The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography
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DOI：
10.1017/s1355838200000303
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发表时间：
2000-05-01
期刊：
影响因子：
4.5
通讯作者：
Noller, HF
中科院分区：
文献类型：
--
作者：
Lancaster, L;Culver, GM;Noller, HF
核糖体蛋白S8是研究最深入的RNA结合蛋白之一，对16S rRNA中心区的组装是必不可少的。为了定位其在核糖体中的周围RNA，我们使用Fe(II)锚定在70年代核糖体蛋白S8表面的9个不同位置，对16S rRNA进行了定向羟基自由基探测。在620茎中经典的S8结合位点附近观察到羟基自由基诱导的切割，并在靠近650、825和860茎的三螺旋连接处观察到其他S8足迹区域的侧翼。此外，在16S rRNA的5‘末端附近、在其5’结构域的300区和在其3‘-主结构域的1070区的切割提供了与其结合不直接相关的RNA元件接近S8的信息。这些数据，以及之前的足迹和交联结果，使蛋白质S8及其周围的RNA元件能够在Thermus thermophilus 70S核糖体的7.8埃图谱中定位。由此产生的模型与之前使用蛋白质-蛋白质和蛋白质-RNA交联、化学和酶足迹以及遗传学进行的大量研究的数据非常一致。
Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-Angstrom map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics.