Identification and characterization of Lon protease as a component of bacterial trans-translation

The trans-translation pathway of bacteria serves to counteract problems associated with interrupted protein synthesis. During trans-translation, a trapped ribosomal complex that is unable to continue or terminate is properly released, the associated mRNA is rapidly degraded, and the incomplete peptide is tagged for targeted proteolysis. Ribosomal rescue helps maintain the pool of functional ribosomes, degradation of the ribosome-stalling mRNA prevents the recurrence of interrupted translation, and degradation of the incomplete peptide limits the cellular burden of abnormal proteins. The bifunctional transfer-messenger RNA (tmRNA) and its dedicated cofactor SmpB protein are essential components of trans-translation. Genetic screens were designed to identify novel components of trans -translation and further characterize this highly conserved pathway. 18,929 Escherichia coli mutants generated by transposon mutagenesis were screened for a specific bacteriophage phenotype associated with cells defective in tmRNA and SmpB, producing 148 primary candidates. Colony PCR analyses of the segment of E. coli genomic DNA containing the genes encoding tmRNA and SmpB suggested that two of the primary candidates contained transposon in this region. To remove mutants that were generally resistant to bacteriophage infection, the primary candidates were screened for sensitivity to a bacteriophage whose development is independent of tmRNA and SmpB function. The secondary bacteriophage screening of the primary candidates left 16 secondary candidates, which were mapped to determine transposon integration sites. Assessment of trans-translation function in the secondary candidates using an endogenous protein tagging assay revealed that each of the three candidates with transposon integrated in the gene encoding Lon protease accumulated excessive levels of the tagged proteins produced during trans-translation. Two reporter assays were optimized specifically for the study of tagged protein turnover in vivo and confirmed that cells defective in Lon protease are unable to efficiently dispose of tagged peptides compared to wild-type cells. In vitro proteolysis experiments using highly purified components showed that Lon preferentially degrades tagged proteins compared to untagged control proteins, thus complementing in vivo experiments. This dissertation discusses the use of genetic screens for the investigation of trans-translation and the experimental course used for the characterization a strong screen candidate.