Detection of tmRNA-mediated trans-translation products in Bacillus subtilis
Detection of tmRNA-mediated trans-translation products in Bacillus subtilis
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DOI：
10.1046/j.1365-2443.2002.00523.x
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发表时间：
2002-03-01
期刊：
影响因子：
2.1
通讯作者：
Muto, A
中科院分区：
文献类型：
--
作者：
Fujihara, A;Tomatsu, H;Muto, A
背景：细菌tmRNA （10Sa RNA）参与反翻译反应，该反应有助于不完全合成肽的降解和停滞核糖体的再循环。为了研究这种反应在枯草芽孢杆菌中的生理作用，我们设计了一个系统来检测受体内陷阱翻译影响的蛋白质。结果：基因组中的野生型tmRNA基因（ssrA）被一个变体ssrA取代，该变体ssrA编码一个标签肽序列，该序列在c端包含6个组氨酸残基（His-tag）和2个天冬氨酸。将细胞内积累的未降解的his标记蛋白用Ni2+-NTA柱和凝胶电泳分离，用抗his标记抗体进行Western blotting检测。结果表明，高温（50℃）比低温（37℃）更容易发生反译。产物的二维凝胶电泳显示了许多不同的斑点，这些斑点代表了反翻译反应的特定靶蛋白。此外，高温和低温培养的细胞产物的二维凝胶模式明显不同。通过产物的n端氨基酸序列鉴定了几种标记蛋白。结论：陷阱翻译在高温下比在低温下发生的频率更高，不同的蛋白质在不同的温度下被标记。
Background: Bacterial tmRNA (10Sa RNA) is involved in a trans-translation reaction, which contributes to the degradation of incompletely synthesized peptides and the recycling of stalled ribosomes. To investigate the physiological roles of this reaction in Bacillus subtilis, we devised a system for detecting the proteins that are subject to in vivo traps-translation.Results: The wild-type tmRNA gene (ssrA) in the genome was replaced by a variant ssrA encoding a tag-peptide sequence containing six histidine residues (His-tag) and two aspartic acids at the C-terminus. The His-tagged proteins that accumulated in the cells without degradation were fractionated by Ni2+-NTA column and gel electrophoresis and were detected by Western blotting with an anti-His-tag antibody. The results showed that the trans-translation occurred more frequently at a high temperature (50 degreesC) than at a low temperature (37 degreesC). Two-dimensional (2D) gel electrophoresis of the products revealed many distinct spots, which represent specific target proteins for the trans-translation reaction. Furthermore, the 2D gel patterns of the products from cells cultured at high and low temperatures were apparently different. Several tagged proteins were identified by the N-terminal amino acid sequences of the products.Conclusion: Traps-translation occurs more frequently at high temperature than at low temperature, and different proteins are tagged at different temperatures.