SsrA-Mediated Tagging in Bacillus subtilis

Abstract

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 ␤-galactosidase fused to an HrcA-controlled promoter. After the predicted proteolysis tag was fused to HrcA, the reporter ␤-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.What happens to translating ribosomes arriving at the 3Ј end of an mRNA molecule lacking a stop codon? Will they fall off (dissociate) or remain stalled? Work carried out over the last 5 years with Escherichia coli has shown that the ribosomes first are stalled and then are released by a novel mechanism designated trans translation (for a recent review, see reference 13). This mechanism requires a small stable RNA (SsrA RNA), also termed 10Sa RNA or tmRNA, which serves both as a tRNA and as an mRNA. When translating ribosomes arrive at the 3Ј end of a truncated mRNA without encountering a stop codon, the process of trans translation is initiated. A tmRNA charged with alanine at its 3Ј end (17, 28) enters the acceptor position of the ribosome and this is followed by transfer of the alanine residue to the nascent polypeptide chain.

kinA mRNA is missing a stop codon in the undomesticated Bacillus subtilis strain ATCC 6051

“…The ssrA gene codes for tmRNA, which possesses both a tRNA-like domain and an mRNA-like coding sequence that together enable tmRNA to function as both a tRNA and an mRNA. This system rescues ribosomes stalled on the mRNAs and directs addition of a proteolysis tag to the C-termini of aberrant protein products, thus targeting them for proteolysis by the Clp protease complex (Gottesman et al, 1998;Wiegert & Schumann, 2001). Thus, the stop-codon-less mRNA of kinA 6051 may be a substrate for tmRNA, leading to rapid degradation of KinA 6051 protein.…”

Proteomic identification of tmRNA substrates

“…Studies in vivo and in vitro suggest that tmRNA substrates are selected by truncation of the mRNA before translation is complete (2). In E. coli, C. crescentus, and B. subtilis, insertion of a strong transcriptional terminator before an in-frame stop codon results in tmRNA tagging of reporter genes (5,27,34,35). In E. coli, tagging can also be induced artificially by depletion of a tRNA or termination factor (36)(37)(38).…”

Self‐compartmentalized bacterial proteases and pathogenesis

“…ClpP can associate with ClpA or ClpX hexamers in Gramnegative bacteria (Kessel et al ., 1995). In Gram-positive bacteria, ClpA is absent but studies suggest that ClpP interacts with Clps C and X instead (Kruger et al ., 2000;Wiegert and Schumann, 2001). There are numerous other Clp ATPases in Gram-positive bacteria (Table 1); however, it is not known exactly how many of them interact with ClpP.…”