Feaga, H. | bioRxiv Science

bioRxiv2024

YebC2 resolves ribosome stalling at polyprolines independent of EF-P and the ABCF ATPase YfmR

Polyproline motifs are essential structural features of many proteins, and recent evidence suggests that EF-P is one of several factors that facilitate their translation. 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 the YebC-family protein YebC2 (formerly YeeI) functions as a translation factor in B. subtilis that resolves ribosome stalling at polyprolines. We demonstrate that YebC2, EF-P and YfmR act independently to support cellular fitness. Moreover, we show that YebC2 interacts directly with the 70S ribosome, supporting a direct role for YebC2 in translation. Finally, we assess the evolutionary relationship between YebC2 and other characterized YebC family proteins, and present evidence that transcription and translation factors within the YebC family have evolved separately. Altogether our work identifies YebC2 as a translation factor that resolves ribosome stalling and provides crucial insight into the relationship between YebC2, EF-P, and YfmR, three factors that prevent ribosome stalling at prolines.

microbiology↗

bioRxiv2019

Ribosome dimerization prevents loss of essential ribosomal proteins during quiescence

The formation of ribosome dimers during periods of quiescence is widespread among bacteria and some higher eukaryotes. However, the mechanistic importance of dimerization is not well understood. In bacteria ribosome dimerization is mediated by the Hibernation Promoting Factor (HPF). Here, we report that HPF from the Gram-positive bacterium Bacillus subtilis preserves active ribosomes by preventing the loss of essential ribosomal proteins. Ribosomes isolated from strains either lacking HPF ({Delta}hpf) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located at the ribosome dimer interface. We used single particle cryo-EM to further characterize ribosomes isolated from a {Delta}hpf mutant strain and observed that many were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function.\n\nSignificance StatementWhen nutrients become scarce, many bacterial species enter an extended state of quiescence. A major challenge of this state is how to attenuate protein synthesis, the most energy consuming cellular process, while preserving ribosomes for the return to favorable conditions. Here, we show that the ribosome-binding protein HPF which dimerizes ribosomes functions to protect essential ribosomal proteins at the dimer interface. HPF is almost universally conserved in bacteria and HPF deletions in diverse species exhibit decreased viability under nutrient limitation. Our data provide mechanistic insight into this phenotype and establish a role for HPF in maintaining translationally competent ribosomes during quiescence.

microbiology↗