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Mol Microbiol. 2010 Nov;78(3):686-700. doi: 10.1111/j.1365-2958.2010.07355.x. Epub 2010 Sep 16.

Importance of the tmRNA system for cell survival when transcription is blocked by DNA-protein cross-links.

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Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.


Anticancer drug 5-azacytidine (aza-C) induces DNA-protein cross-links (DPCs) between cytosine methyltransferase and DNA as the drug inhibits methylation. We found that mutants defective in the tmRNA translational quality control system are hypersensitive to aza-C. Hypersensitivity requires expression of active methyltransferase, indicating the importance of DPC formation. Furthermore, the tmRNA pathway is activated upon aza-C treatment in cells expressing methyltransferase, resulting in increased levels of SsrA tagged proteins. These results argue that the tmRNA pathway clears stalled ribosome-mRNA complexes generated after transcriptional blockage by aza-C-induced DPCs. In support, an ssrA mutant is also hypersensitive to streptolydigin, which blocks RNA polymerase elongation by a different mechanism. The tmRNA pathway is thought to act only on ribosomes containing a 3' RNA end near the A site, and the known pathway for releasing RNA 3' ends from a blocked polymerase involves Mfd helicase. However, an mfd knockout mutant is not hypersensitive to either aza-C-induced DPC formation or streptolydigin, indicating that Mfd is not involved. Transcription termination factor Rho is also likely not involved, because the Rho-specific inhibitor bicyclomycin failed to show synergism with either aza-C or streptolydigin. Based on these findings, we discuss models for how E. coli processes transcription/translation complexes blocked at DPCs.

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