Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20
Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20
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DOI：
10.1017/s1355838298981201
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发表时间：
1998-12-01
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：
Culver, GM;Noller, HF
利用直接羟基自由基探针对核糖体蛋白S20的16S核糖体RNA邻域进行了定位，包括30S亚基和70S核糖体。在S20的14、23、49和57位引入半胱氨酸残基，用于1-(对溴乙酰氨基苯甲基)-Fe(11)-EDTA的连接。用Fe(11)衍生的S20与剩余的小亚基核糖体蛋白和S63核糖体RNA(RRNA)一起进行体外重组，得到了具有功能的30S亚基。纯化了含有Fe(11)-S20的30S亚基和70S核糖体，并从系留的Fe(11)中产生了羟基自由基。16S rRNA骨架的羟基自由基裂解通过引物延伸进行监测。四个位置的Fe(11)在16S rRNA中都有不同的切割模式，而在30S和70S核糖体中这些模式没有显著差异。将切割位点定位到5‘结构域的160-200、320和340-350位置，以及16S rRNA倒数第二茎远端的1427-1430和1439-1458位置，将这些区域在三维上彼此靠近。这些结果与先前将320个16S rRNA元件定位在这些16S rRNA元件附近的足迹数据一致，提供了S20和S17一样位于30S亚基底部附近的证据。
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(ll)-EDTA. In vitro reconstitution using Fe(ll)-derivatized S20, together with the remaining small subunit ribosomal proteins and S63 ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(ll)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(ll). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(ll) 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 320 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.