The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5 0 side of the A residue in the nucleotide sequence 5 0 -UA(A/C)-3 0 of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2 0 :3 0 -cyclic phosphate group and a free 5 0 -OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2 0 -OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.

Due to its long half-life compared to messenger RNA, bacterial transfer RNA is known as stable RNA. Here, we show that tRNAs become highly unstable as part of Escherichia coli's response to amino acid starvation. Degradation of the majority of cellular tRNA occurs within twenty minutes of the onset of starvation for each of several amino acids. Both the non-cognate and cognate tRNA for the amino acid that the cell is starving for are degraded, and both charged and uncharged tRNA species are affected. The alarmone ppGpp orchestrates the stringent response to amino acid starvation. However, tRNA degradation occurs in a ppGpp-independent manner, as it occurs with similar kinetics in a relaxed mutant. Further, we also observe rapid tRNA degradation in response to rifampicin treatment, which does not induce the stringent response. We propose a unifying model for these observations, in which the surplus tRNA is degraded whenever the demand for protein synthesis is reduced. Thus, the tRNA pool is a highly regulated, dynamic entity. We propose that degradation of surplus tRNA could function to reduce mistranslation in the stressed cell, because it would reduce competition between cognate and near-cognate charged tRNAs at the ribosomal A-site.

The bacterial parD toxin-antitoxin system of plasmid R1 encodes two proteins, the Kid toxin and its cognate antitoxin, Kis. Kid cleaves RNA and inhibits protein synthesis and cell growth in Escherichia coli. Here, we show that Kid promotes RNA degradation and inhibition of protein synthesis in rabbit reticulocyte lysates. These new activities of the Kid toxin were counteracted by the Kis antitoxin and were not displayed by the KidR85W variant, which is nontoxic in E. coli. Moreover, while Kid cleaved single-and double-stranded RNA with a preference for UAA or UAC triplets, KidR85W maintained this sequence preference but hardly cleaved double-stranded RNA. Kid was formerly shown to inhibit DNA replication of the ColE1 plasmid. Here we provide in vitro evidence that Kid cleaves the ColE1 RNA II primer, which is required for the initiation of ColE1 replication. In contrast, KidR85W did not affect the stability of RNA II, nor did it inhibit the in vitro replication of ColE1. Thus, the endoribonuclease and the cytotoxic and DNA replication-inhibitory activities of Kid seem tightly correlated. We propose that the spectrum of action of this toxin extends beyond the sole inhibition of protein synthesis to control a broad range of RNA-regulated cellular processes.

The mazEF (chpA) toxin-antitoxin system of Escherichia coli is involved in the cell response to nutritional and antibiotic stresses as well as in bacterial-programmed cell death. Valuable information on the MazF toxin was derived from the determination of the crystal structure of the MazE/MazF complex and from in vivo data, suggesting that MazF promoted ribosome-dependent cleavage of messenger RNA. However, it was concluded from recent in vitro analyses using a MazF-(His6) fusion protein that MazF was an endoribonuclease that cleaved messenger RNA specifically at 5 0 -ACA-3 0 sites situated in singlestranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5 0 side of residue A in 5 0 -NAC-3 0 sequences (where N is preferentially U or A). MazFdependent cleavage occurred at target sequences situated either in single-or double-stranded RNA regions. These activities were neutralized by a His6-MazE antitoxin. Although essentially consistent with previous in vivo reports on the substrate specificity of MazF, our results strongly suggest that the endoribonuclease activity of MazF may be modulated by additional factors to cleave messenger and other cellular RNAs.

Toxin–antitoxin systems, as found in bacterial plasmids and their host chromosomes, play a role in the maintenance of genetic information, as well as in the response to stress. We describe the basic biology of the parD/kiskid toxin–antitoxin system of Escherichia coli plasmid R1, with an emphasis on regulation, toxin activity, potential applications in biotechnology and its relationships with related toxin–antitoxin systems. Special reference is given to the ccd toxin–antitoxin system of plasmid F because its toxin shares structural homology with the toxin of the parD system. Inter‐relations with related toxin–antitoxin systems present in the E. coli chromosome, such as the parD homologues chpA/mazEF and chpB and the relBE system, are also reviewed. The combined structural and functional information that is now available on all these systems, as well as the ongoing controversy regarding the role of the chromosomal toxin–antitoxin loci, have made this review especially timely.