The ABC-F protein EttA gates ribosome entry into the translation elongation cycle

Abstract

ABC-F proteins have evaded functional characterization even though they comprise one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein Energy-dependent Translational Throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies, including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistent with this inference, ΔettA cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a novel mechanism sensitive to cellular energy status.

The ABCs of the ribosome

“…An ~70-residue ‘linker’ motif, unique to ABC-F proteins lies between the tandem NBDs and contains an extended α-helix. Overall, the protein adopts an ‘open’ conformation, with the subdomains organized loosely with respect to one another, as expected for an ABC protein without a bound nucleotide 1 .…”

“…Two new reports describe the discovery of a bacterial protein factor, energy-dependent translation throttle A (EttA), which can regulate translation in response to changes in cellular energy homeostasis 1 , 2 . The ability of EttA to link protein synthesis to changing metabolite levels opens up the exciting possibility that other translation factors might also respond to different physiological cues, as is the case for transcriptional regulation.…”

“…1 report the X-ray crystal structure of nucleotide-free EttA at 2.4-Å resolution ,and Chen et al 2 report the cryo-EM structure of a mutant version of EttA bound to the ribosome. This EttA mutant (E188Q E470Q, termed EttA-EQ 2 ) is a key tool in the present studies because it is able to bind, but not hydrolyze, ATP.…”

“…This EttA mutant (E188Q E470Q, termed EttA-EQ 2 ) is a key tool in the present studies because it is able to bind, but not hydrolyze, ATP. In the crystal lattice, EttA forms a domain-swapped dimer, although several observations, including the cryo-EM reconstitution, suggest that the monomer is the biologically active form 1 , 2 . The tandem nucleotide-binding domains (NBDs) of EttA resemble those of other ABC proteins except for two insertions termed the ‘toe’ and ‘arm', the latter being a longer extension comprising two α-helices.…”

Detailed Mutational Analysis of Vga(A) Interdomain Linker: Implication for Antibiotic Resistance Specificity and Mechanism

“…It has been suggested that ARE proteins might interact with the ribosome and affect the binding of antibiotics to their target site (17). Very recently, the function of the first ABC-F protein, EttA, as a translation factor which regulates protein synthesis depending on cell energy status has been characterized (19,20). The similarity of Vga(A)* to EttA (31% identity) allows us to envisage a mechanism of Vga(A) action based on the EttA one, providing a more-detailed insight into the probable involvement of Vga(A) in antibiotic resistance by ribosome protection than ever before.…”