Importance of the tmRNA system for cell survival when transcription is blocked by DNA-protein cross-links.
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.
Kuo
H Kenny
HK
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Krasich
Rachel
R
Bhagwat
Ashok S
AS
Kreuzer
Kenneth N
KN
D001374:Azacitidine / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050296:Microbial Viability / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C096222:tmRNA; D001374:Azacitidine
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