Directed hydroxyl radical probing of 16S rRNA in the ribosome: spatial proximity of RNA elements of the 3' and 5' domains.
Directed hydroxyl radical probing of 16S rRNA in the ribosome: spatial proximity of RNA elements of the 3' and 5' domains.
复制标题
核糖体中 16S rRNA 的定向羟基自由基探测：3 和 5 结构域的 RNA 元件的空间邻近性。
DOI：
10.1017/s1355838299990192
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Noller,HF
中科院分区：
文献类型：
--
作者：
Newcomb,LF;Noller,HF
我们之前已经证明，从拴在RNA内特定位点的Fe（II）中直接探测16S rRNA的羟基自由基可以提供有关70S核糖体中RNA - RNA接近性的有价值信息。在这里，我们扩展了这项研究，并提出了nt 424在16S rRNA中的探测数据。为了将Fe（II）拴在rRNA中的位置424上，我们通过体外转录将RNA作为两个分别对应于nt 1-423和424 - 1542的片段，在RNA中创建了一个特定的不连续。Fe(II)-BABE在424位共价连接到5 ' -鸟苷-α-硫代酸，并由这两条rRNA和小亚基蛋白重构30S亚基。通过分离70S核糖体，选择了能够与50S亚基结合的重组30S亚基。由束缚的Fe（II）原位产生的羟基自由基，在三维空间上切割了RNA主链中靠近Fe（II）的位置，并通过引物延伸确定了切割位置。拴在位置424上的Fe（II）诱导16S rRNA 5 ‘结构域nt 424、513和531周围以及16S核糖体RNA 3 ’结构域nt 1008、1029、1044和1208周围的切割。这些数据限制了420、1015、1030和1000/1040螺旋的位置，这些螺旋的结构信息很少。由于16S rRNA的5 ' -和3 ' -结构域分别构成30S亚基的体和头，因此这些发现为RNA元件在30S的体和头中的接近性提供了直接证据。
We have shown previously that directed hydroxyl radical probing of 16S rRNA from Fe(II) tethered to specific sites within the RNA gives valuable information about RNA–RNA proximities in 70S ribosomes. Here, we extend that study and present probing data from nt 424 in 16S rRNA. To tether an Fe(II) to position 424 in the rRNA we created a specific discontinuity in the RNA by in vitro transcription of the RNA as two separate fragments corresponding to nt 1–423 and 424–1542. An Fe(II)-BABE was covalently attached to a 5′-guanosine-α-phosphorothioate at position 424 and 30S subunits were reconstituted from the two pieces of rRNA and the small subunit proteins. Reconstituted 30S subunits capable of associating with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved positions in the RNA backbone that were close in three-dimensional space to the Fe(II), and the sites of cleavage were identified using primer extension. Fe(II) tethered to position 424 induces cleavage around nt 424, 513, and 531 in the 5′-domain of 16S rRNA and around nt 1008, 1029, 1044, and 1208 in the 3′-domain of 16S ribosomal RNA. These data constrain the positions of the 420, 1015, 1030 and 1000/1040 helices, for which there is little structural information. Since the 5′- and 3′-domains of 16S rRNA constitute the body and head, respectively, of 30S subunits, these findings provide direct evidence for proximity of RNA elements in the body and head of 30S.