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

Abstract

SummaryAnticancer drug 5-azacytidine (aza-C) induces DNAprotein 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-Cinduced 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 3Ј RNA end near the A site, and the known pathway for releasing RNA 3Ј 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.

Functions that protect Escherichia coli from DNA–protein crosslinks

“…Several previous reports have characterized E. coli aza-C hypersensitive mutants, leading to the conclusions above about excision and recombinational repair (7–10,16,32). Additional mutants that have been shown to be hypersensitive include lexA , recG , ruvABC , priA , priB , polA , uvrD, ssrA, smpB, hflC and dnaJ (7–9,16,32). A limitation of some of these past studies of aza-C-hypersensitive mutants is that they relied on the endogenous Dcm protein for DPC formation, leaving some uncertainty about whether hypersensitivity was indeed due to DPC formation or some other effect of aza-C on cell metabolism.…”

“…The transposon mutagenesis screen utilized strain HK21 carrying plasmid pR215 (16). HK21 is a derivative of strain ER1793 (obtained from New England Biolabs) and has the following genotype: F − fhuA2 Δ(lacZ)r1 glnV44 e14 − (McrA − ) trp-31 his-1 rpsL104 xyl-7 mtl-2 metB1 Δ(mcrC-mrr)114::IS10) ΔsulA (Keio deletion) dinD::lacZ.…”

“…Plasmids pR215 and pBAD-MEcoRII were used to express M.EcoRII (16). The pR215 plasmid is a pACYC184-derived plasmid containing the tetracycline resistance marker and the M.EcoRII gene under control of its own promoter (34).…”

“…Both E. coli and phage T7 RNA polymerases have been shown to stall at DPC sites, although in the latter case a very inefficient and mutagenic read through was also documented (12,15). Indirect evidence for inhibition of transcription in vivo comes from the finding that aza-C-induced DPCs trigger tagging by the tmRNA system, which releases and thereby recycles ribosomes that are stalled or have reached a premature RNA end (16) (also see below). tmRNA functions by binding to the empty A-site of a stalled ribosome and inducing the ribosome to translate the mRNA coding sequence of tmRNA.…”

“…It should be noted that while the recA dcm mutant is still hypersensitive to aza-C, overexpression of a cytosine methyltransferase greatly increases the sensitivity of a recA knockout, clearly showing an involvement of RecA in some process related to DPC formation (9). In addition, a subset of the above mutants ( recB , recG , ssrA , smpB , hflC and dnaJ ) were shown to have DPC-dependent sensitivity to aza-C (8,9,16), and our approach in the current work explicitly demands DPC-dependent sensitivity.…”