Type I toxin-antitoxin systems encoded on bacterial chromosomes became the focus of research during the past years. However, little is known in terms of structural requirements, kinetics of interaction with their targets and regulatory mechanisms of the antitoxin RNAs. Here, we present a combined in vitro and in vivo analysis of the bsrG/SR4 type I toxin-antitoxin system from Bacillus subtilis. The secondary structures of SR4 and bsrG mRNA and of the SR4/bsrG RNA complex were determined, apparent binding rate constants calculated and functional segments required for complex formation narrowed down. The initial contact between SR4 and its target was shown to involve the SR4 terminator loop and loop 3 of bsrG mRNA. Additionally, a contribution of the stem of SR4 stem-loop 3 to target binding was found. On SR4/bsrG complex formation, a 4 bp double-stranded region sequestering the bsrG Shine Dalgarno (SD) sequence was extended to 8 bp. Experimental evidence was obtained that this extended region caused translation inhibition of bsrG mRNA. Therefore, we conclude that SR4 does not only promote degradation of the toxin mRNA but also additionally inhibit its translation. This is the first case of a dual-acting antitoxin RNA.
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