Brimacombe R, Mitchell P, Osswald M, Stade K, Bochkariov D
Max-Planck-Institut für Molekulare Genetik, Berlin, Dahlem, Germany.
FASEB J. 1993 Jan;7(1):161-7. doi: 10.1096/fasebj.7.1.8422963.
An aryl trifluoromethyl diazirine photoreactive derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(IArg) and this derivatized tRNA was bound to Escherichia coli 70S ribosomes. After irradiation at 350 nm the site of cross-linking to the 16S RNA was analyzed by our standard procedures and found to lie within the secondary structural element comprising bases 956-983; this region contains two modified nucleotides at positions 966 and 967. Similarly, an aryl azido photoreactive derivative was attached to the phenylalanine residue of Phe-tRNA(Phe), and the derivatized aminoacyl tRNA was bound to the ribosome either at the A- or the P-site. In both cases, after irradiation at 250 nm, the cross-link site was localized to position 2439 of the 23S RNA; in the secondary structure of the latter the neighboring nucleotide 2442 is base-paired to a modified nucleotide at position 2069. Taken together with other cross-linking data, these results now directly implicate a total of 27 out of the 29 modified nucleotides in E. coli 16S and 23S RNA as lying within or close to the functional center of the ribosome.
将一种芳基三氟甲基重氮丙啶光反应性衍生物连接到tRNA（IArg）32位的2-硫代胞苷残基上，然后将这种衍生化的tRNA与大肠杆菌70S核糖体结合。在350nm波长下照射后，通过我们的标准程序分析与16S RNA的交联位点，发现其位于包含956 - 983位碱基的二级结构元件内；该区域在966位和967位含有两个修饰核苷酸。同样，将一种芳基叠氮光反应性衍生物连接到苯丙氨酰 - tRNA（Phe）的苯丙氨酸残基上，然后将这种衍生化的氨酰tRNA在A位点或P位点与核糖体结合。在这两种情况下，在250nm波长下照射后，交联位点都定位在23S RNA的2439位；在后者的二级结构中，相邻核苷酸2442与2069位的一个修饰核苷酸形成碱基对。结合其他交联数据，这些结果现在直接表明大肠杆菌16S和23S RNA的29个修饰核苷酸中有27个位于核糖体功能中心内或其附近。
