Heilek G M, Marusak R, Meares C F, Noller H F
Sinsheimer Laboratories, University of California, Santa Cruz 95064.
Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1113-6. doi: 10.1073/pnas.92.4.1113.
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-Cys31]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.
局部羟基自由基探测已被用于探索大肠杆菌30S核糖体亚基结构中一个独特位置周围的rRNA邻域。通过试剂1-(对溴乙酰氨基苄基)-EDTA将Fe(II)连接到核糖体蛋白S4的Cys-31上。然后将[Fe-Cys31]S4与16S rRNA复合，或通过与16S rRNA和其余30S亚基蛋白混合物进行体外重组，将其掺入活性30S核糖体亚基中。由连接的Fe产生的羟基自由基导致16S rRNA链在其5'结构域的两个局部区域发生切割。一个区域跨越位置419 - 432，靠近先前通过化学和酶保护（足迹法）以及交联研究确定为S4的RNA结合位点的多螺旋连接点。第二个定向切割位点包括核苷酸297 - 303，该位点与一个免受蛋白质S16化学修饰的位点重叠，S16是核糖体中S4的近邻。这些结果提供了有关16S rRNA三维组织的有用信息，并表明其5'结构域的这两个区域在空间上与蛋白S4的Cys-31紧密相邻。
