Detection of tmRNA‐mediated trans‐translation products in Bacillus subtilis

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Abstract
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 trans‐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 °C) than at a low temperature (37 °C). 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: Trans‐translation occurs more frequently at high temperature than at low temperature, and different proteins are tagged at different temperatures.