SsrA-mediated tagging in Bacillus subtilis.
A general mechanism in bacteria to rescue stalled ribosomes involves a stable RNA encoded by the ssrA gene. This RNA, termed tmRNA, encodes a proteolytic peptide tag which is cotranslationally added to truncated polypeptides, thereby targeting them for rapid proteolysis. To study this ssrA-mediated mechanism in Bacillus subtilis, a bipartite detection system was constructed that was composed of the HrcA transcriptional repressor and the bgaB reporter gene coding for a heat-stable beta-galactosidase fused to an HrcA-controlled promoter. After the predicted proteolysis tag was fused to HrcA, the reporter beta-galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA. Replacement of the two C-terminal alanine residues of the tag by aspartate rendered the repressor stable. Replacement of the hrcA stop codon by a transcriptional terminator sequence rendered the protein unstable; this was caused by trans translational addition of the proteolytic tag. Inactivating the B. subtilis ssrA or smpB (yvaI) gene prevented the trans translational tagging reaction. Various protease-deficient strains of B. subtilis were tested for proteolysis of tagged HrcA. HrcA remained stable only in clpX or clpP knockouts, which suggests that this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in B. subtilis.
Wiegert
T
T
Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany.
Schumann
W
W
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D018388:Codon, Terminator; D004268:DNA-Binding Proteins; D049071:Endopeptidase Clp; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012329:RNA, Bacterial / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism
D001426:Bacterial Proteins; D018388:Codon, Terminator; D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C430328:small protein B; C096222:tmRNA; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases