B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria.

B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains and reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation by the ribosome quality control pathway. Notably, we see no evidence of mRNA cleavage by MutS2, nor does it promote ribosome rescue by tmRNA as SmrB cleavage does in E. coli. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in various bacteria.

Inhibition of SRP-dependent protein secretion by the bacterial alarmone (p)ppGpp.

The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli.