Department of Structural Biology and Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
Nucleic Acids Res. 2010 Jul;38(13):e143. doi: 10.1093/nar/gkq390. Epub 2010 May 25.
Site-specific labeling of Escherichia coli ribosomes has allowed application of single-molecule fluorescence spectroscopy and force methods to probe the mechanism of translation. To apply these approaches to eukaryotic translation, eukaryotic ribosomes must be specifically labeled with fluorescent labels and molecular handles. Here, we describe preparation and labeling of the small and large yeast ribosomal subunits. Phylogenetically variable hairpin loops in ribosomal RNA are mutated to allow hybridization of oligonucleotides to mutant ribosomes. We demonstrate specific labeling of the ribosomal subunits, and their use in single-molecule fluorescence and force experiments.
大肠杆菌核糖体的定点标记使单分子荧光光谱和力方法能够应用于探测翻译机制。为了将这些方法应用于真核翻译，必须用荧光标记物和分子手柄特异性标记真核核糖体。在这里，我们描述了小亚基和大亚基酵母核糖体的制备和标记。核糖体 RNA 中进化上可变的发夹环被突变，以允许寡核苷酸与突变核糖体杂交。我们证明了核糖体亚基的特异性标记，并将其用于单分子荧光和力实验。
