Gulle H, Hoppe E, Osswald M, Greuer B, Brimacombe R, Stöffler G
Max-Plank-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, FRG.
Nucleic Acids Res. 1988 Feb 11;16(3):815-32. doi: 10.1093/nar/16.3.815.
RNA-protein cross-links were introduced into E. coli 50S ribosomal subunits by treatment with 2-iminothiolane followed by mild ultraviolet irradiation. After partial digestion of the RNA, the cross-linked RNA-protein complexes were separated by our recently published three-step procedure. In cases where this separation was inadequate, a further purification step was introduced, involving affinity chromatography with antibodies to the ribosomal 50S proteins. Analysis of the isolated complexes enabled four new cross-link sites on the 23S RNA to be identified, as well as re-confirming several previously established sites. The new sites are as follows: Protein L2 is cross-linked within an oligonucleotide at positions 1818-1823 in the 23S RNA, protein L4 within positions 320-325, protein L24 within positions 99-107, and protein L27 within positions 2320-2323.
通过用2-亚氨基硫杂环戊烷处理，随后进行温和的紫外线照射，将RNA-蛋白质交联引入大肠杆菌50S核糖体亚基。在RNA进行部分消化后，通过我们最近发表的三步程序分离交联的RNA-蛋白质复合物。在这种分离不充分的情况下，引入了进一步的纯化步骤，包括用针对核糖体50S蛋白质的抗体进行亲和色谱。对分离出的复合物的分析使得能够鉴定出23S RNA上的四个新交联位点，并再次确认了几个先前确定的位点。新位点如下：蛋白质L2在23S RNA中1818 - 1823位的寡核苷酸内交联，蛋白质L4在320 - 325位内交联，蛋白质L24在99 - 107位内交联，蛋白质L27在2320 - 2323位内交联。
