Interference of mRNA Function by Sequence-specific Endoribonuclease PemK

Abstract

In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin. The pemI-pemK system is an addiction module present on plasmid R100. It helps to maintain the plasmid by post-segregational killing in E. coli population. Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E. coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis. Further studies reveal that PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the endoribonuclease activity of PemK. PemK cleaves only single-stranded RNA preferentially at the 5 or 3 side of the A residue in the "UAH" sequences (where H is C, A, or U). Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E. coli. The pemK homologue genes have been identified on the genomes of a wide range of bacteria. We propose that PemK and its homologues form a novel endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.In Escherichia coli, programmed cell death is proposed to be mediated through the system called "addiction module," which consists of a pair of genes encoding a toxin and an antitoxin (1). The addiction module has the following properties. (a) The toxic protein is stable, whereas the antitoxin is a labile protein. (b) The toxin and the antitoxin are coexpressed from an operon and interact with each other to form a stable complex. (c) Their expression is auto-regulated either by the toxin-antitoxin complex or by the antitoxin alone. When the co-expression is inhibited under stress conditions, the antitoxin is degraded by proteases, enabling the toxin to act on its target. In E. coli, some extrachromosomal elements are known to contain addiction modules causing the bacterial programmed cell death by the so-called postsegregational killing effect. The most studied extrachromosomal addiction modules are the phd-doc system on bacteriophage P1 (2-5), the ccdA-ccdB system on factor F (6 -9), the kis-kid system on plasmid R1 (10 -13), and the pemIpemK system on plasmid R100 (14 -17). Interestingly, the E. coli chromosome also contains several addiction module systems, such as the relBE system (18 -21), the mazEF system (22-25), and the chpB system (26 -28).The cellular effects of the toxins in the addiction modules have been studied quite extensively. CcdB, the toxin in the ccdA-ccdB system, interacts with DNA gyrase to block DNA replication (7,29), and RelE, the toxin in the relBE system, is not able to degrade free RNA but cleaves mRNA in the ribosome A site with high codon specificity (21). Recently, it was demonstrated that the A-site mRNA cleavage can occur in the absence of RelE (30). The exact mechanism of the A-site mRNA cleavage is still unknown. It has been proposed that MazF (ChpAK)...

Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit

“…8). This result was in striking contrast to all other TA toxins-MazF, PemK, ChpBK, RelE, and HigB-known to target and rapidly degrade mRNA (27,28,30,31). Therefore, we suspected that the presence of Doc might stabilize mRNA.…”

Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot

“…Previous results suggested a general mRNA interferase function for ToxN Pa ; however, this activity was not shown directly, and ToxN Pa cleaved housekeeping gene transcripts in vitro even though it was present in complex with its antitoxin (14). In contrast, studies of mRNA interferases from type II TA systems showed that the addition of the antitoxin in a 1:1 stoichiometry completely inhibits the toxin's activity in vitro (16)(17)(18). To confirm the mechanism of ToxN Pa toxicity, Northern blots of the highly expressed housekeeping genes ompA, dksA, and lpp were performed following overexpression of ToxN Pa and the subsequent co-overexpression of ToxI Pa .…”

Biochemical characterization of a chromosomal toxin–antitoxin system in Mycobacterium tuberculosis

“…Another DNA fragment containing a T7 promoter followed by the E. coli era gene was obtained by PCR amplification with the T7 primer 5 0 -AGATCTCGATCCCGCAAATTAAT-3 0 and the primer era-2 5 0 -TTAAAGATCGTCAACGTAACCG-3 0 with pET28a-Era plasmid [12] as template. The Rv0707 mRNA and era mRNA were prepared from these two DNA fragments respectively with the RiboMAXä T7 large scale RNA production system (Promega).…”