EttA regulates translation by binding the ribosomal E site and restricting ribosome-tRNA dynamics

Abstract

Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, Energy-dependent Translational Throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we demonstrate that the ATP-bound form of EttA binds to the ribosomal tRNA exit (E) site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA binding (P) site. Using single-molecule fluorescence resonance energy transfer (smFRET), we show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, where the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors.

The ABCs of the ribosome

“…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.…”

“…EttA-EQ 2 binds the exit (E) site, filling the space between the L1 stalk and P-site tRNA (Fig. 1) 2 . A model of EttA in the ‘closed’ conformation best fits the cryo-EM density, consistent with the EttA-EQ2 variant being trapped in the ATP-bound state.…”

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.…”