Abstract
Type II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.
Highlights
Toxin-antitoxin systems are widespread in prokaryotes, with many studies focused on the Type II systems comprised of a protein toxin and a tightly interacting but labile protein a ntitoxin[1,2,3,4]
Toxins in the Rel-superfamily are grouped based on structural homology and include sub-families denoted as HigB, YafQ, YoeB, and RelE; toxins in each of these sub-families cleave mRNA within the ribosomal A-site, the specific catalytic mechanisms are d ivergent[18,19,20,21,22,23]
We have recently identified a YoeB toxin from Agrobacterium tumefaciens, referred to as AtYoeB, that is a ribosomal-independent mRNase consistent with some previously characterized YoeB t oxins[21,26,27]
Summary
Toxin-antitoxin systems are widespread in prokaryotes, with many studies focused on the Type II systems comprised of a protein toxin and a tightly interacting but labile protein a ntitoxin[1,2,3,4]. We have recently identified a YoeB toxin from Agrobacterium tumefaciens, referred to as AtYoeB, that is a ribosomal-independent mRNase consistent with some previously characterized YoeB t oxins[21,26,27] During these studies with AtYoeB we noted an additional catalytic activity, the cleavage of DNA, which is not previously characterized for this protein family or fold. The dimeric AtYoeB toxin interacts with double-strand DNA yielding two independent binding events with affinities (KD) in the micromolar range. These results highlight that this fold is able to function as a non-specific nuclease, with the ribosome improving the efficiency of catalysis while restricting the substrate to mRNA
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