Multiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the four Escherichia coli K12 ABCFs - EttA, Uup, YbiT, and YheS - using assays previously employed to demonstrate EttA gates the first step of polypeptide elongation on the ribosome dependent on ATP/ADP ratio. A Δ uup knockout, like Δ ettA , exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither Δ ybiT nor Δ yheS exhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based on in vitro translation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ ₂ ) that trap them in the ATP-bound conformation. These variants all strongly stabilize the same global conformational state of a ribosomal elongation complex harboring deacylated tRNA ^Val in the P site. However, EQ ₂ -Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ ₂ -YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ ₂ -EttA and EQ ₂ -YbiT fully inhibit in vitro translation of an mRNA encoding luciferase, while EQ ₂ -Uup and EQ ₂ -YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ ₂ -Uup or EQ ₂ -YheS, while EQ ₂ -YbiT inhibits synthesis of both peptide bonds and EQ ₂ -EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the four E. coli ABCF paralogs all having different activities on translating ribosomes, and they suggest that there remains a substantial amount of functionally uncharacterized "dark matter" involved in mRNA translation.

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