Background: Bacterial transfer-messenger RNA (tmRNA, 10Sa RNA) is involved in a trans-translation reaction which contributes to the degradation of incompletely synthesized peptides and to the recycling of stalled ribosomes. However, its physiological role in the cell remains elusive. In this study, an ef®cient system for controlling the expression of the gene for tmRNA (ssrA), as well as a tmRNA gene-defective strain (ssrA::cat), were constructed in Bacillus subtilis. The effects of tmRNA on the growth of the cells were investigated under various physiological culture conditions using these strains.

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.

SummarySpore formation in Bacillus subtilis is significantly impaired by the deletion of the gene for tmRNA (ssrA), which facilitates the trans-translation reaction that rescues stalled ribosomes and degrades incompletely synthesized peptides. Microscopic analysis revealed that the sporulation of most DssrA cells is blocked after forespore formation. Expression analysis of lacZ-fused genes directed by several RNA polymerase s factors showed that the synthesis of active s K , encoded by the sigK gene, is predominantly inhibited in DssrA cells. The defect in s K synthesis is attributable to a defect in the skin element excision, which generates the sigK gene, caused in turn by reduced expression of SpoIVCA (recombinase) in DssrA cells.

TreP [trehalose-permease (phosphotransferase system (PTS) trehalose-specific enzyme IIBC component)] is one of the target proteins of tmRNA-mediated trans-translation in Bacillus subtilis [Fujihara et al. (2002) Detection of tmRNA-mediated trans-translation products in Bacillus subtilis. Genes Cells, 7, 343-350]. The TreP synthesis is subject to CcpA-dependent carbon catabolite repression (CCR), and the treP gene contains catabolite-responsive element (cre) sequence, a binding site of repressor protein CcpA, in the coding region. Here, we demonstrated that the tmRNA-tagging of TreP occurs depending on the gene for CcpA. In the presence of CcpA, the transcription of treP mRNA terminates at 8-9 nucleotides upstream of the 5'-edge of the internal cre sequence, and translational switch to the tag-sequence occurs at the 101st amino-acid (asparagine) position from N-terminus of TreP. The results show that trans-translation reaction is involved in the tagging and degradation of the N-terminal TreP fragment produced by truncated mRNA, which is a product of transcriptional roadblock by CcpA binding to the cre sequence in the internal coding region.