Abstract
EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. A detailed understanding of its mechanism of action is limited due to the lack of high-resolution structures along its ATPase cycle. Here we present the cryo-electron microscopy (cryo-EM) structures of EttA from Mycobacterium tuberculosis (Mtb), referred to as MtbEttA, in complex with the Mtb 70S ribosome initiation complex (70SIC) at the pre-hydrolysis (ADPNP) and transition (ADP-VO4) states, and the crystal structure of MtbEttA alone in the post-hydrolysis (ADP) state. We observe that MtbEttA binds the E-site of the Mtb 70SIC, remodeling the P-site tRNA and the ribosomal intersubunit bridge B7a during the ribosomal ratcheting. In return, the rotation of the 30S causes conformational changes in MtbEttA, forcing the two nucleotide-binding sites (NBSs) to alternate to engage each ADPNP in the pre-hydrolysis states, followed by complete engagements of both ADP-VO4 molecules in the ATP-hydrolysis transition states. In the post-hydrolysis state, the conserved ATP-hydrolysis motifs of MtbEttA dissociate from both ADP molecules, leaving two nucleotide-binding domains (NBDs) in an open conformation. These structures reveal a dynamic interplay between MtbEttA and the Mtb ribosome, providing insights into the mechanism of translational regulation by EttA-like proteins.
Highlights
EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle
Order to understand the conformational change of MtbEttA at the post-hydrolysis state compared to the previous step of the ATPhydrolysis, we focused on half of the domainswapped dimer composed of the NBD1 from protomer A, and NBD2 from protomer B, which was analyzed in the same fashion for the crystal structure of the nucleotide-free EcoEttA12
Using cryo-electron microscopy (cryo-EM) and X-ray crystallography, we have determined the structures of MtbEttA and its interaction with the Mycobacterium tuberculosis (Mtb) ribosome in the course of ATP hydrolysis
Summary
EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. In the post-hydrolysis state, the conserved ATP-hydrolysis motifs of MtbEttA dissociate from both ADP molecules, leaving two nucleotide-binding domains (NBDs) in an open conformation. These structures reveal a dynamic interplay between MtbEttA and the Mtb ribosome, providing insights into the mechanism of translational regulation by EttA-like proteins. The first structurally determined ABCF protein, EttA from Escherichia coli (referred to as EcoEttA), was found to bind to the ribosome and “throttle” (or regulate) its entry into the translation elongation cycle, depending on the ATP/ADP ratio[12,13]. Like other members in the ABC superfamily, most ABC-F proteins are ATPases with highly conserved ATP binding/hydrolysis motifs, albeit with variations of the aromatic residue at the A-loop within NBD112
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