The tmRNA system is essential when transcription is blocked by DNA-protein crosslinks H. Kenny Kuo1, Ashok S. Bhagwat2 and Kenneth N. Kreuzer1,* Abstract Anticancer drug 5-azacytidine (aza-C) induces crosslinks between cytosine methyltransferase and DNA as the drug inhibits the methylation reaction. We found that mutants defective in the tmRNA translational quality control system are hypersensitive to aza-C. Hypersensitivity of these mutants requires expression of active methyltransferase, indicating that hypersensitivity is dependent on DNA-methyltransferase crosslink formation. Furthermore, the tmRNA pathway is activated upon aza-C treatment in cells expressing methyltransferase, resulting in increased SsrA tagging of cellular proteins. These results support a “chain-reaction” model, in which transcription complexes blocked by aza-C-induced DNA-protein crosslinks result in ribosomes stalling on the attached nascent transcripts, and the tmRNA pathway is invoked for cleaning up the resulting pile-ups. In support of this model, an ssrA mutant is also hypersensitive to antibiotic streptolydigin, which blocks RNA polymerase elongation. These results reveal a novel role for the tmRNA system in clearance of coupled transcription/translation complexes in which RNA polymerase has become blocked.