Culver G M, Noller H F
Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
RNA. 1998 Dec;4(12):1471-80. doi: 10.1017/s1355838298981201.
The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160-200, 320, and 340-350 in the 5' domain, and to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.
利用定向羟基自由基探测技术，对核糖体蛋白S20在30S亚基和70S核糖体中的16S核糖体RNA邻域进行了图谱绘制。在S20的第14、23、49和57位氨基酸处引入了半胱氨酸残基，并用于连接1-(对溴乙酰氨基苄基)-Fe(II)-EDTA。使用Fe(II)衍生化的S20与其余小亚基核糖体蛋白和16S核糖体RNA（rRNA）进行体外重组，得到了功能性的30S亚基。纯化了含有Fe(II)-S20的30S亚基和70S核糖体，并从连接的Fe(II)产生羟基自由基。通过引物延伸监测16S rRNA主链的羟基自由基切割。从连接到四个位置中每个位置的Fe(II)观察到16S rRNA中不同的切割模式，并且这些模式在30S和70S核糖体中没有显著差异。切割位点被定位到5'结构域中的160 - 200、320和340 - 350位，以及16S rRNA倒数第二个茎的远端的1427 - 1430和1439 - 1458位，使这些区域在三维空间中彼此靠近。这些结果与先前将S20定位在这些16S rRNA元件附近的足迹数据一致，提供了证据表明S20与S17一样位于30S亚基的底部附近。
