Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.
Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.
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DOI：
10.1017/s1355838298981134
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发表时间：
1998-11
期刊：
影响因子：
4.5
通讯作者：
Dmitri Mundus;P. Wollenzien
中科院分区：
文献类型：
--
作者：
Dmitri Mundus;P. Wollenzien
利用位点特异性光交联技术研究了大肠杆菌30S亚基中16S rRNA位置U788/ U789的RNA邻域。在这些研究中，位点特异性补骨脂素（SSP）在17a侧链上含有一个巯基，首先通过退火和光转移的互补引导DNA添加到核苷酸U788/U789上。从DNA和未修饰的RNA中纯化修饰RNA。在一些实验中，通常在8 A距离交联的SSP与叠氮苯酰溴（APAB）发生衍生物化，产生距补骨脂素（SSP25APA） 4'位置最多25A的光反应性叠氮基部分。重组含有SSP、SSP25APA或对照16S rRNA的16S rRNA，分离30S颗粒。含有SSP或SSP25APA的重组亚基具有正常的蛋白质组成，在tRNA结合中具有活性，除了G791的酮醛反应性增加和其他四个区域的轻微变化外，具有通常的化学反应性模式。在活化缓冲液中辐照衍生化的30S亚基产生了多个分子内RNA交联，这些交联通过凝胶电泳显示和分离，并通过引物延伸进行表征。SSP试剂产生的四个主要交联位点分别位于U561/U562、U920/U921、C866和U723；当使用SSP25APA试剂时，在G693处发现了第五个主要交联。一些额外的交联频率较低，特别是与APA试剂。这些数据表明，在活化的30S亚基中，核苷酸U788/U789的中心位置靠近解码区和中心假结。
Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.