DIRECTED HYDROXYL RADICAL PROBING OF 16S RIBOSOMAL-RNA USING FE(II) TETHERED TO RIBOSOMAL-PROTEIN S4
DIRECTED HYDROXYL RADICAL PROBING OF 16S RIBOSOMAL-RNA USING FE(II) TETHERED TO RIBOSOMAL-PROTEIN S4
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DOI：
10.1073/pnas.92.4.1113
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发表时间：
1995-02-14
影响因子：
11.1
通讯作者：
NOLLER, HF
中科院分区：
文献类型：
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作者：
HEILEK, GM;MARUSAK, R;NOLLER, HF
局部羟基自由基探测已被用来探索周围的一个独特的位置在大肠杆菌30 S核糖体亚基的结构rRNA邻居。Fe（II）通过试剂1-（p-bromoacetamidobenzyl）-EDTA连接到核糖体蛋白S4的Cys-31。然后[Fe-Cys（31）] S4与16 S rRNA复合，或通过与16 S rRNA和剩余30 S亚基蛋白的混合物体外重构而掺入活性30 S核糖体亚基中。由拴系的Fe产生的羟基自由基导致16 S rRNA链在其5'结构域的两个局部区域中裂解。一个区域跨越位置419-432，靠近之前通过化学和酶保护（足迹法）和交联研究置于S4 RNA结合位点的多螺旋连接。定向切割的第二个位点包括核苷酸297-303，其与被蛋白质S16（核糖体中S4的近邻）保护免于化学修饰的位点重叠。这些结果提供了有关16 S rRNA三维结构的有用信息，并表明其5'结构域的这两个区域与蛋白S4的Cys-31在空间上非常接近。
Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit. Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA. [Fe-Cys(31)] S4 was then complexed with 16S rRNA or-incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain. One region spans positions 419-432 and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies. A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome. These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.