Lon protease degrades transfer-messenger RNA-tagged proteins

Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with lambda-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.     

Involvement of Clp protease activity in modulating the Bacillus subtilissigmaw stress response

The induction of Bacillus subtilis genes controlled by the extracytoplasmic function alternative sigma factor sigmaW is strongly impaired in a strain deleted for the ClpP peptidase gene and in a double knockout of the ClpX and ClpE ATPase genes. Truncated soluble forms of the sigmaW anti-sigma factor RsiW are stabilized in a clpP minus strain as revealed by the green fluorescent reporter protein fused to the N-terminus of RsiW and by pulse-chase experiments. Conserved alanine residues are present in the transmembrane region of RsiW, and mutations in these positions abolish induction of sigmaW-controlled genes. Following alkaline shock, a truncated cytoplasmic form of RsiW is detectable in a strain expressing a triple alanine mutant allele of rsiW. These data point to a mechanism where the trans-membrane segment of RsiW contains a cryptic proteolytic tag that is uncovered as a result of intramembrane proteolysis of RsiW by RasP (YluC). After RasP-clipped RsiW is detached from the membrane, this proteolytic tag becomes crucial for the complete degradation of RsiW by cytoplasmic proteases and the release of sigmaW. ClpXP plays a major role in this third proteolytic step of stress-induced degradation of RsiW. Overexpression of SsrA-tagged green fluorescent protein as a ClpXP substrate protein reduces alkali induction of a sigmaW-controlled gene by a factor of about three, indicating that a titration mechanism is able to tune the sigmaW-mediated stress response to the cellular state.     

tmRNA-dependent trans-translation is required for sporulation in Bacillus subtilis

Spore 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 Δ ssrA cells is blocked after forespore formation. Expression analysis of lacZ -fused genes directed by several RNA polymerase σ factors showed that the synthesis of active σ^K, encoded by the sigK gene, is predominantly inhibited in Δ ssrA cells. The defect in σ^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 Δ ssrA cells.     

LambdaN-GFP: an RNA reporter system for live-cell imaging

We describe a GFP-based RNA reporter system (lambdaN-GFP) to visualize RNA molecules in live mammalian cells. It consists of GFP fused to an arginine-rich peptide derived from the phage lambda N protein, lambdaN22, which binds a unique minimal RNA motif and can be used to tag any RNA molecule. LambdaN-GFP uses a small and easy to engineer RNA tag, reducing the likelihood of perturbing the function of the tagged RNA molecule.     

Identification of a helix-turn-helix motif of Bacillus thermoglucosidasius HrcA essential for binding to the CIRCE element and thermostability of the HrcA-CIRCE complex,indicating a role as a thermosensor

In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in B. thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B. thermoglucosidasius, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B. thermoglucosidasius and B. subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.     

Dynamics of substrate denaturation and translocation by the ClpXP degradation machine

ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.     

Proteolytic adaptor for transfer-messenger RNA-tagged proteins from alpha-proteobacteria

We have identified an analog of SspB, the proteolytic adaptor for transfer-messenger RNA (tmRNA)-tagged proteins, in Caulobacter crescentus. C. crescentus SspB shares limited sequence similarity with Escherichia coli SspB but binds the tmRNA tag in vitro and is required for optimal proteolysis of tagged proteins in vivo.     

Phenotypes conferred by the Bacillus subtilis recM13 mutation and the din23 fusion.

The din23 fusion encodes a B. subtilis SOS-inducible regulatory region fused to the E. coli lacZ gene (Love et al., 1985). A strain encoding the din23 fusion and a recM13 allele showed low-level constitutive beta-galactosidase expression, was induced for beta-galactosidase production by DNA gyrase inhibitors but not by DNA-damaging agents, and was slightly induced by a variety of agents which do not normally induce the SOS regulon. The din23 fusion itself resulted in high levels of spontaneous prophage induction in wild-type, recM- and recA-hosts, despite the fact that the din23recM13 strain was not induced for beta-galactosidase production by DNA-damaging agents. The results suggest that the recM gene may be involved with the regulation of the RecA protease-mediated SOS response, while the din23 gene may be involved with the regulation of an alternative function which results in the cleavage of prophage repressor.