YfmR is a translation factor that prevents ribosome stalling and cell death in the absence of EF-P

Abstract

Protein synthesis is catalyzed by the ribosome and a host of highly conserved elongation factors. Most elongation factors that are conserved in all domains of life are essential, such as EF-Tu (e/aEF1A) and EF-G (e/aEF2). In contrast, the universally conserved elongation factor P (EF-P/eIF5A) is essential in eukaryotes but is dispensable in most bacteria. EF-P prevents ribosome stalling at difficult-to translate sequences, especially polyprolines. Since efp deletion phenotypes range from modest to lethal in different bacterial species, we hypothesized that some bacteria encode an uncharacterized elongation factor with compensatory functions. To identify this factor, we used Tn-seq to screen for genes that are essential in the absence of EF-P in Bacillus subtilis. This screen identified YfmR, a member of the ABCF family of ATPases, as a translation factor that is essential when efp is deleted. We find that depleting YfmR from ∆efp cells decreases actively translating ribosomes and increases free ribosomal subunits bound to initiator tRNA, suggesting that ribosomes stall in early elongation. We also find that deleting efp from the spore-forming pathogen Bacillus anthracis causes a severe survival defect in liquid culture and in the presence of macrophages. B. anthracis ∆efp also does not produce detectable spores. We find that heterologous expression of B. subtilis YfmR in B. anthracis ∆efp cells partially rescues the severe growth and sporulation defects of this mutant. Our results therefore identify YfmR as an important translation factor that becomes essential in the absence of EF-P, and suggest that the essential function of YfmR and EF-P is to assist translation during early elongation.

The ABCF proteins in Escherichia coli individually cope with ‘hard-to-translate’ nascent peptide sequences

“…However, the E. coli strain lacking all four ABCFs exhibited susceptibility to drugs related to translation elongation ( Supplementary Figure S9C ). In addition, synthetic growth defect was observed between pth ts mutant and ettA or ybiT (Figure 4E ), as well as between efp and uup (Figure 6C , the identical phenotype in Gram-positive bacteria was reported in a recently published paper and a preprint: 63 , 64 ). These demonstrate that each ABCF collectively contributes to maintaining the robustness of translation elongation.…”

EF-P and its paralog EfpL (YeiP) differentially control translation of proline-containing sequences

“…The importance to alleviate ribosome stalling at consecutive prolines is further underlined by the existence of additional rescue systems namely the ATP-Binding Cassette family-F (ABCF) protein Uup in E. coli and its ortholog YfmR in B. subtilis 37,[59][60][61] . In interplay with EF-P, Uup/YfmR and EfpL can facilitate translation of XP(P)X containing proteins.…”

RqcH supports survival in the absence of non-stop ribosome rescue factors

“…CRISPR interference. To create the sgRNA targeting SmpB, we amplified pJMP2 using primer sp60 (29) and reverse primer HRH175 (43). The Phusion PCR product was DpN-1 treated for 2.5hrs at 37˚C followed by PNK treatment for 1h.…”