Döring T, Mitchell P, Osswald M, Bochkariov D, Brimacombe R
Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.
EMBO J. 1994 Jun 1;13(11):2677-85. doi: 10.1002/j.1460-2075.1994.tb06558.x.
A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.
一种光反应性重氮丙啶衍生物连接到来自大肠杆菌的tRNA(Arg)I第32位的2-硫代胞苷残基上。这种修饰的tRNA在合适的条件下与大肠杆菌核糖体的A、P或E位点结合。重氮丙啶标签经光激活后，通过我们的标准程序确定与16S rRNA的交联位点。三个tRNA结合位点中的每一个都显示出特征性的交联模式。对于A位点的tRNA，观察到与16S RNA的1378位有一个主要交联，与936位有一个次要交联。对于P位点，与693位以及957和/或966位有主要交联，与1338位有一个次要交联。E位点结合的tRNA显示与693位（与P位点相同）以及1376/1378位有主要交联（与从A位点观察到的交联相似但不完全相同）。对伴随交联的核糖体蛋白的免疫分析表明，S7是来自所有三个tRNA位点交联的主要靶点，S11是次要产物。根据30S核糖体亚基解码区域的整体拓扑结构对结果进行了讨论。
