title: Lon protease degrades transfer-messenger RNA-tagged proteins.
abstract: 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.
authors.affiliation: Department of Biochemistry and Cell Biology, Center for Infectious Diseases, Stony Brook University, Stony Brook, NY 11794, USA. akarzai@stonybrook.edu
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Choy
authors.forename: Latt Latt
authors.initials: LL
authors.lastname: Aung
authors.forename: A Wali
authors.initials: AW
authors.lastname: Karzai
mesh_terms: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D016254:Mutagenesis, Insertional; D010957:Plasmids; D049070:Protease La / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C096222:tmRNA; D012343:RNA, Transfer; D049070:Protease La