Powers T, Noller H F
Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
RNA. 1995 Apr;1(2):194-209.
Complexes between 16S rRNA and purified ribosomal proteins, either singly or in combination, were assembled in vitro and probed with hydroxyl radicals generated from free Fe(II)-EDTA. The broad specificity of hydroxyl radicals for attack at the ribose moiety in both single- and double-stranded contexts permitted probing of nearly all of the nucleotides in the 16S rRNA chain. Specific protection of localized regions of the RNA was observed in response to assembly of most of the ribosomal proteins. The locations of the protected regions were in good general agreement with the footprints previously reported for base-specific chemical probes, and with sites of RNA-protein crosslinking. New information was obtained about interaction of ribosomal proteins with 16S rRNA, especially with helical elements of the RNA. In some cases, 5' or 3' stagger in the protection pattern on complementary strands suggests interaction of proteins with the major or minor groove, respectively, of the RNA. These results reinforce and extend previous data on the localization of ribosomal proteins with respect to structural features of 16S rRNA, and offer many new constraints for three-dimensional modeling of the 30S ribosomal subunit.
16S rRNA与纯化的核糖体蛋白单独或组合形成的复合物在体外组装，并用游离Fe(II)-EDTA产生的羟基自由基进行探测。羟基自由基对单链和双链环境中核糖部分攻击的广泛特异性使得能够探测16S rRNA链中几乎所有的核苷酸。响应于大多数核糖体蛋白的组装，观察到RNA局部区域的特异性保护。受保护区域的位置与先前报道的碱基特异性化学探针的足迹以及RNA-蛋白质交联位点总体上高度一致。获得了关于核糖体蛋白与16S rRNA相互作用的新信息，特别是与RNA螺旋元件的相互作用。在某些情况下，互补链上保护模式的5'或3'交错分别表明蛋白质与RNA的大沟或小沟相互作用。这些结果加强并扩展了先前关于核糖体蛋白相对于16S rRNA结构特征定位的数据，并为30S核糖体亚基的三维建模提供了许多新的限制条件。
