The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.
The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.
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tRNA 中央折叠的核糖体邻域：从位于 A、P 或 E 位点的 tRNA 47 位开始的交联。
DOI：
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发表时间：
1995
影响因子：
14.9
通讯作者：
R. Brimacombe
中科院分区：
文献类型：
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作者：
M. Osswald;T. Döring;R. Brimacombe
在核糖体A、P或E位点特有的条件下，用二氮杂衍生物修饰来自大肠杆菌tRNA(Phe)第47位的天然核苷酸3-(3-氨基-3-羧丙基)尿苷(Acp3U)，并在合适的mRNA类似物存在下与核糖体结合。在350 nm处光激活后，通过我们的标准程序鉴定与核糖体蛋白质和RNA的交联链。30S亚基蛋白S19(弱S9和S13)是A位tRNA的交联靶蛋白，P位是S7、S9和S13亚基，E位是S7亚基。同样，在50年代，亚基L16和L27来自A位，L1、L5、L16、L27和L33来自P位，L1和L33来自E位。在16S rRNA中，位于P和E位点的687和727位之间的位置为1318和1350(P位)，1350和1387位(E位)；23S rRNA中位于A位的865和910位之间，1845和1892位(P位)，1892和1945位(A位)，2282和2358位(P位)，2242和2461位(P位)，2461和2488位(A位)，2488和2539位(均为A位)和2572和2603位(A和P位)。在大多数(但不是所有)情况下，通过引物延伸分析可以更精确地定位交联点。
The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.