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

Abstract

Bacterial small RNAs perform numerous regulatory roles, including acting as antitoxic components in toxin-antitoxin systems. In type III toxin-antitoxin systems, small processed RNAs directly antagonize their toxin protein partners, and in the systems characterized the toxin and antitoxin components together form a trimeric assembly. In the present study, we sought to define how the RNA antitoxin, ToxI, inhibits its potentially lethal protein partner, ToxN. We show through cross-inhibition experiments with the ToxIN systems from Pectobacterium atrosepticum (ToxIN Pa ) and Bacillus thuringiensis (ToxIN Bt ) that ToxI RNAs are highly selective enzyme inhibitors. Both systems have an "addictive" plasmid maintenance phenotype. We demonstrate that ToxI Pa can inhibit ToxN Pa in vitro both in its processed form and as a repetitive precursor RNA, and this inhibition is linked to the self-assembly of the trimeric complex. Inhibition and self-assembly are both mediated entirely by the ToxI Pa RNA, with no requirement for cellular factors or exogenous energy. Finally, we explain the origins of ToxI antitoxin selectivity through our crystal structure of the ToxIN Bt complex. Our results show how a processed RNA pseudoknot can inhibit a deleterious protein with exquisite molecular specificity and how these self-contained and addictive RNA-protein pairs can confer different adaptive benefits in their bacterial hosts.RNA inhibition | mRNA interferase | RNA-protein complex | abortive infection | plasmid stabilization

Mutational Analysis of the Antitoxin in the Lactococcal Type III Toxin-Antitoxin System AbiQ

“…Our 5= RACE experiments showed that the cleavage site was located within the repeat between adenine 26 and adenine 27 in an adenine-rich region (A/AAA), one nucleotide away from a predicted in silico ABIQantiQ cleavage site (/AAAA) (43). Interestingly, the toxin of the two other type III TA systems also cleaved RNA fragments in adenine-rich regions (ToxIN Pa , AA/AU; ToxIN Bt , A/AAAA) (32). In antiQ, this polyadenine sequence is found in the complete repeats (R1 and R2) and also in the last 0.8 repeat (R3).…”

“…However, when the protein (ToxN) is expressed in trans, only 1.5 repeats are necessary (25). Presumably, the key would be to have at least one complete mature repeat fragment (from cleavage site to cleavage site), with this fragment playing a critical role in toxin regulation within the type III TA systems (28,32).…”

“…It also has been shown that the secondary structure (pseudoknot) of ToxI RNA is essential for the antitoxic activity (28). Under stress conditions, ToxN is free and can target essential mRNAs, leading to cell growth arrest and preventing phage replication (25,28,32).…”

“…Only a few type III TA systems have been characterized to date (17,25,28,32). ToxIN, an Abi system from Pectobacterium atrosepticum (ToxIN Pa ), was the first to be studied and is the model for type III systems (25).…”

Effect of the Abortive Infection Mechanism and Type III Toxin/Antitoxin System AbiQ on the Lytic Cycle of Lactococcus lactis Phages

“…In type III TA systems, the antitoxin molecule forms a pseudoknot structure of three antitoxin repetitions bound to three toxin molecules, leading to a hetero-hexamer triangular structure (55,56). It has also been demonstrated that the free toxin can cleave, through its endoribonuclease activity, the cognate antitoxins (36,54) as well as housekeeping bacterial RNA molecules (55,56), leading to cell death. During the phage infection process, this TA interaction is likely disrupted, leading to cell death and abortion of the phage infection.…”