Comparative genetic, biochemical, and biophysical analyses of the fourE. coliABCF paralogs support distinct functions related to mRNA translation

Abstract

AbstractMultiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the fourEscherichia coliK12 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 Δuupknockout, like ΔettA, exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither ΔybiTnor ΔyheSexhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based onin vitrotranslation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ2) 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 tRNAValin the P site. However, EQ2-Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ2-YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ2-EttA and EQ2-YbiT fully inhibitin vitrotranslation of an mRNA encoding luciferase, while EQ2-Uup and EQ2-YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ2-Uup or EQ2-YheS, while EQ2-YbiT inhibits synthesis of both peptide bonds and EQ2-EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the fourE. coliABCF 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.