Detection of tmRNA-mediated trans-translation products in Bacillus subtilis.
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 degrees C) than at a low temperature (37 degrees 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.


CONCLUSIONS
Trans-translation occurs more frequently at high temperature than at low temperature, and different proteins are tagged at different temperatures.
Fujihara
Ai
A
Department of Biology, Faculty of Science, Hirosaki University, Hirosaki 036-8561, Japan.
Tomatsu
Hisashi
H
Inagaki
Sachi
S
Tadaki
Toshimasa
T
Ushida
Chisato
C
Himeno
Hyouta
H
Muto
Akira
A
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D001426:Bacterial Proteins; D010455:Peptides; D012329:RNA, Bacterial; C096222:tmRNA