The translation of mRNAs without in-frame stop codons results in the following problems: the ribosomes are stalled and sequestered on the defective mRNA, the incomplete polypeptides produced are potentially toxic to the cells, and the defective mRNA can result in more futile translational cycles. Bacteria have developed a unique system, called trans-translation to resolve all of the above-mentioned problems. The key players of the trans-translation system are tmRNA and its protein cofactor SmpB. One part of my work showed that the system mediates the degradation of non-stop mRNAs by recruiting RNase R to stalled ribosomes through the interaction between the C-terminal lysine-rich domain of RNase R and the trans-translation machinery. In addition, the SmpB-tmRNA system adds a peptide tag to the C-termini of the nascent products of mRNAs lacking an in-frame stop codon, marking them for proteolysis. The other part of my work showed that in Escherichia coli, in addition to the ClpXP system, Lon protease also degrades tmRNA-tagged proteins, but with much lower efficiency. I then studied a unique case in Mycoplasma pneumoniae (Mp) where Lon is the primary protease degrading the tmRNA proteins due to the absence of Clp family proteases. I identified two discrete signaling motifs in the Mp-tmRNA tag for Lon recognition. I also showed evidence that the Mp-Lon and Mp-tmRNA tag have co-evolved to allow enhanced Lon binding to the tag and more efficient degradation of the Mp-tmRNA tagged proteins. The discrepancy in the substrate specificity between Ec-Lon and Mp-Lon promoted us to further explore the general substrate specificity of Lon in difference bacterial species. I was also interested in the role of Lon protease in the pathogenesis of Yersinia pestis. I performed a genome wide profiling of Lon substrates and identified novel substrates, many of which are master regulators of gene expression in Yersinia. I also identified and compared the preferred cleavage sites of Lon protease in multiple substrates. We are now working on deciphering the biological significance of the degradation of these substrates by Lon, with the hope of shedding more light on the role Lon is playing in Yersinia pathogenesis.