SsrA-mediated tagging in Bacillus subtilis

A general mechanism in bacteria to rescue stalled ribosomes involves a stab le RNA encoded by the ssrA gene. This RNA, termed tmRNA, encodes a proteoly tic 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 constructe d that was composed of the HrcA transcriptional repressor and the bgaB repo rter gene coding for a heat-stable beta -galactosidase fused to an HrcA-con trolled promoter. After the predicted proteolysis tag was fused to HrcA, th e reporter beta -galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA, Replacement of the two C-termin al 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 addi tion of the proteolytic tag. Inactivating the B, subtilis ssrA or smpB (yva I) gene prevented the trans translational tagging reaction. Various proteas e-deficient strains of S, subtilis were tested for proteolysis of tagged Hr cA, HrcA remained stable only in clpX or clpP knockouts, which suggests tha t this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in B, subtilis.