LAUSR

Like other forms of life, bacteria utilize a variety of mechanisms to survive stress. Some of these mechanisms mitigate the physiological impact of the stress, whereas others enable cells to simply wait out the stressful condition. Among the cellular mediators of the latter response are toxin-antitoxin (TA) systems [1]. When activated in response to stress, TA systems cause bacterial cells to enter a dormant state in which growth ceases and their metabolic needs are greatly diminished. Subsequently, when more favorable conditions return, the cells reawaken and resume growth. Mechanisms of this kind have also been implicated in antibiotic tolerance, in which a small fraction of an otherwise susceptible bacterial population manages to survive exposure to an antibiotic by hibernation, repopulating the host when the antibiotic treatment ends [2]. Toxin-antitoxin systems are widespread throughout the bacterial realm, where almost all species encode several of them in discrete genetic modules from which a toxin and its cognate antitoxin are both expressed [3,4]. Among the most common are those that comprise a ribonuclease (toxin) and a protein inhibitor (antitoxin) that sequesters the ribonuclease in a catalytically inactive complex [5]. Upon exposure to stress, selective degradation of the antitoxin unleashes the toxin, whose catalytic action forces the cell into a dormant state. Some toxins of this kind (e.g., MazF, ChpB, MqsR, and HicA in Escherichia coli) are sequence-specific endonucleases that choose their RNA targets rather indiscriminately [6–9]. Others (e.g., RelE, YoeB, HigB, YafO, and YafQ in E. coli) are activated by ribosome binding, which causes them to preferentially target ribosome-associated transcripts [10–12]. By selectively degrading easily replenishable mRNAs and avoiding rRNAs and tRNA, toxins of the latter variety cause less damage, which in principle should make it easier for cell growth to resume once the stress ends. On the basis of bioinformatic analysis, Lupas and coworkers proposed several years ago that the E. coli prlF-yhaV operon encodes another such TA system conserved in many proteobacterial species [13]. They went on to show that YhaV is indeed a ribonuclease that ordinarily is inhibited by complex formation with PrlF but can arrest cell growth when produced in excess of its antitoxin [14]. Unexpectedly, despite statistically significant sequence similarity to RelE, purified YhaV appeared capable of degrading RNA in the absence of ribosomes. In this issue of FEBS Letters, Choi et al. revisit the question of whether the activity of YhaV is ribosomeindependent and present multiple lines of contrary evidence indicating that its endonuclease activity is in fact potentiated by ribosome binding [15]. In their hands, purified E. coli YhaV cleaves RNA only in the presence of ribosomes. Its activation appears to be specific for bacterial ribosomes, as it is able to abort mRNA translation in vitro when added to a cell-free system derived from E. coli but not one derived from rabbit reticulocytes. Immunoblot analysis of polysomes extracted from E. coli and fractionated on sucrose gradients reveals that YhaV cosediments with 70S ribosomes and 50S ribosomal subunits, consistent with an affinity for E. coli ribosomes. Furthermore, when selectively overproduced in E. coli, YhaV rapidly degrades mRNA, including transcripts that are normally long-lived, but leaves ribosomal RNA unscathed. It cleaves these mRNAs within the proteincoding region, usually between codons and with little apparent sequence specificity, providing further evidence that its activity is dependent on translation. What then to make of the previous report that YhaV does not require ribosomes for its endonuclease activity [14]? Significantly, that investigation did not test whether the activity observed for purified YhaV could be stimulated by ribosomes; nor was the target specificity of the toxin examined in vivo. Furthermore, both that study and the current inquiry by Choi et al. were complicated by the need to isolate the toxin as a complex with its neutralizing antitoxin, free it from the complex by denaturation, and then refold it in the absence of the antitoxin in order to examine the endonuclease activity of the purified toxin in vitro. The