Strains, plasmids, and primers.

Ribosome stalling is a major source of cellular stress. Therefore, many specialized elongation factors help prevent ribosome stalling. One of the best characterized of these factors is EF-P, which prevents ribosome stalling at polyproline tracts and other difficult-to-translate sequences. Recent evidence suggests that other factors also facilitate translation of polyproline motifs. For example, YfmR was recently identified as a protein that prevents ribosome stalling at proline-containing sequences in the absence of EF-P. Here, we show that YebC2 (formerly YeeI) functions as a translation factor in Bacillus subtilis that resolves ribosome stalling at polyprolines. YebC2 associates with the ribosome, supporting a direct role for YebC2 in translation. Moreover, YebC2 can reduce ribosome stalling and support cellular fitness in the absence of EF-P and YfmR. Finally, we present evidence that YebC2 is evolutionarily distinct from previously characterized YebC-family transcription factors and demonstrate that these paralogs have distinct physiological roles in B. subtilis. Altogether our work identifies YebC2 as a translation factor that resolves ribosome stalling in B. subtilis and provides crucial insight into the relationship between YebC2, EF-P, and YfmR, three factors that prevent ribosome stalling at polyprolines.