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Detection of tmRNA‐mediated trans‐translation products in Bacillus subtilis
Author(s) -
Fujihara Ai,
Tomatsu Hisashi,
Inagaki Sachi,
Tadaki Toshimasa,
Ushida Chisato,
Himeno Hyouta,
Muto Akira
Publication year - 2002
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.2002.00523.x
Subject(s) - biology , bacillus subtilis , translation (biology) , gel electrophoresis , rna , ribosome , biochemistry , microbiology and biotechnology , histidine , amino acid , gene , messenger rna , genetics , bacteria
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 Ni 2+ ‐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.