Newcomb L F, Noller H F
Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
Biochemistry. 1999 Jan 19;38(3):945-51. doi: 10.1021/bi981644a.
Directed hydroxyl radical probing of 16S ribosomal RNA from Fe(II) tethered to specific sites within the RNA was used to determine RNA-RNA proximities in 70S ribosomes. We have transcribed 16S ribosomal RNA in vitro as two separate fragments, covalently attached an Fe(II) probe to a 5'-guanosine-alpha-phosphorothioate at the junction between the two fragments, and reconstituted 30S subunits with the two separate pieces of RNA and the small subunit proteins. Reconstituted 30S subunits capable of association with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved sites in the 16S rRNA backbone that were close in three-dimensional space to the Fe(II), and a primer extension was used to identify these sites of cleavage. Two sets of 16S ribosomal RNA fragments, 1-360/361-1542 and 1-448/449-1542, were reconstituted into active 30S subunits. Fe(II) tethered to position 361 results in cleavage of 16S rRNA around nucleotides 34, 160, 497, 512, 520, 537, 552, and 615, as well as around positions 1410, 1422, 1480, and 1490. Fe(II) tethered to position 449 induces cleavage around nucleotide 488 and around positions 42 and 617. Fe(II) tethered to the 5' end of 16S rRNA induces cleavage of the rRNA around nucleotides 5, 601, 615, and 642. These results provide constraints for the positioning of these regions of 16S rRNA, for which there has previously been only limited structural information, within the 30S subunit.
利用连接到RNA内特定位点的Fe(II)对16S核糖体RNA进行定向羟基自由基探测，以确定70S核糖体中RNA - RNA的接近程度。我们在体外将16S核糖体RNA转录为两个单独的片段，在两个片段之间的连接处将Fe(II)探针共价连接到5'-鸟苷-α-硫代磷酸酯上，并用这两个单独的RNA片段和小亚基蛋白重建30S亚基。通过分离70S核糖体来选择能够与50S亚基结合的重建30S亚基。由连接的Fe(II)原位产生的羟基自由基会切割16S rRNA主链中在三维空间上与Fe(II)接近的位点，并使用引物延伸来鉴定这些切割位点。两组16S核糖体RNA片段，即1 - 360/361 - 1542和1 - 448/449 - 1542，被重建为有活性的30S亚基。连接到位置361的Fe(II)会导致16S rRNA在核苷酸34、160、497、512、520、537、552和615周围以及位置1410、1422、1480和1490周围发生切割。连接到位置449的Fe(II)会诱导在核苷酸488以及位置42和617周围发生切割。连接到16S rRNA 5'端的Fe(II)会诱导rRNA在核苷酸5、601、615和642周围发生切割。这些结果为16S rRNA这些区域在30S亚基内的定位提供了限制条件，而此前关于这些区域的结构信息非常有限。
