Lieberman K R, Noller H F
Center for the Molecular Biology of RNA, University of California, Santa Cruz, CA, 95064, USA.
J Mol Biol. 1998 Dec 18;284(5):1367-78. doi: 10.1006/jmbi.1998.2236.
L15, a 15 kDa protein of the large ribosomal subunit, interacts with over ten other proteins during 50 S assembly in vitro. We have probed the interaction L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting, and have used localized hydroxyl radical probing, generated from Fe(II) tethered to unique sites of L15, to characterize the three-dimensional 23 S rRNA environment of L15. Footprinting of L15 was done by reconstituting purified, recombinant L15 with core particles derived from Escherichia coli 50 S subunits by treatment with 2 M LiCl. The cores migrate as compact 50 S-like particles in sucrose gradients, contain 23 S and 5 S rRNA, and lack a subset of the 50 S proteins, including L15. Using both Fe(II).EDTA and dimethyl sulfate, we have identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23 S rRNA. This footprint cannot be detected when L15 is incubated with "naked" 23 S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle.Protein-tethered hydroxyl radical probing was done using mutants of L15 containing single cysteine residues at amino acid positions 68, 71 and 115. The mutant proteins were derivatized with 1-[p-(bromo-acetamido)benzyl]-EDTA. Fe(II), bound to core particles, and hydroxyl radical cleavage was initiated. Distinct but overlapping sets of cleavages were obtained in the footprinted region of domain II, and in specific regions of domains I, IV and V of 23 S rRNA. These data locate L15 in proximity to several 23 S rRNA elements that are dispersed in the secondary structure, consistent with its central role in the latter stages of 50 S subunit assembly. Furthermore, these results indicate the proximity of these rRNA regions to one another, providing constraints on the tertiary folding of 23 S rRNA.
L15是一种存在于大核糖体亚基中的15 kDa蛋白质，在体外50 S亚基组装过程中与其他十多种蛋白质相互作用。我们通过化学足迹法研究了L15与50 S核糖体亚基中23 S rRNA的相互作用，并利用连接到L15特定位点的Fe(II)产生的局部羟基自由基探测法，来表征L15周围23 S rRNA的三维环境。L15的足迹分析是通过用2 M LiCl处理从大肠杆菌50 S亚基衍生的核心颗粒，再与纯化的重组L15重组来完成的。这些核心颗粒在蔗糖梯度中以紧密的50 S样颗粒形式迁移，包含23 S和5 S rRNA，并且缺少包括L15在内的一部分50 S蛋白质。使用Fe(II).EDTA和硫酸二甲酯，我们在23 S rRNA结构域II中跨越核苷酸572 - 654的区域确定了一个强烈的L15足迹。当L15与“裸露的”23 S rRNA孵育时，无法检测到这个足迹，这表明L15结合位点的形成需要一个部分组装的颗粒。蛋白质连接的羟基自由基探测是使用在氨基酸位置68、71和115含有单个半胱氨酸残基的L15突变体进行的。突变蛋白用1 - [对-(溴乙酰氨基)苄基]-EDTA进行衍生化。与核心颗粒结合的Fe(II)引发羟基自由基切割。在结构域II的足迹区域以及23 S rRNA的结构域I、IV和V的特定区域获得了不同但重叠的切割组。这些数据表明L15靠近几个分散在二级结构中的23 S rRNA元件，这与其在50 S亚基组装后期的核心作用一致。此外，这些结果表明这些rRNA区域彼此接近，为23 S rRNA的三级折叠提供了限制条件。
