Featured Article - October 2014
Short description: Complementary structural, genetic, biochemical and biophysical approaches identify a new translation regulator that responds to changing ATP/ADP ratios.
ATP-binding cassette (ABC) proteins are one of the most widely distributed protein superfamilies. The ABC-F subfamily is composed of soluble proteins that have been implicated in a variety of processes. The Hunt (PSI NESG), Frank and Gonzalez groups chose to study an Escherichia coli ABC-F protein, YjjK, because its motifs resemble those of other ABC proteins, eEF3 and ABCE1/RLI1, previously reported to interact with the ribosome. After their analyses, YjjK is renamed EttA (energy-dependent translational throttle A).
The 2.4-Å resolution crystal structure of nucleotide-free EttA (PDB 4FIN) confirmed that its tandem ABC domains, like other ABC-F proteins, are separated by an 81-aa linker; however, each ABC domain contains an additional insertion. The ABC domains adopt an open configuration with accessible ATP-binding sites, and modeling suggests that a single rigid-body movement would bring the ABCs together in an ATP-sandwich configuration.
The authors used a mutant defective in ATP binding and hydrolysis, EttA-EQ2, for functional analyses. This mutation inhibits translation in vivo and slows cell growth, so its effect on translation was studied in greater detail. For translation initiation, a tRNA-fMet binds to the P site along with an appropriately charged tRNA in the A site, and formation of the first peptide bond transfers fMet onto the amino acid of the A-site tRNA. Subsequent ribosomal translocation localizes the now empty tRNA to the E site. In an in vitro translation system, EttA-EQ2 trapped the ribosome after the formation of the first peptide bond, with the dipeptide remaining attached to the tRNA in the A site. EttA had little effect when this initiation step had transitioned to elongation (i.e., after tripeptide formation).
For the cryo-electron microscopy reconstruction, the authors used EttA-EQ2 to bind an initiating ribosome complex. Density attributable to EttA-EQ2 was seen at the E site. Notably, only one state of the initiating ribosome was observed, supporting the idea that EttA could block translocation. Structure fitting and modeling identified regions of the ribosome and P-site tRNA-fMet contacted by EttA. Single-molecule analyses further showed that EttA inhibits both ribosome and tRNA dynamics.
But what makes EttA an “energy-dependent throttle”? While analysis of wild-type and mutant enzymes showed that ATP hydrolysis is required to displace EttA from the E site, EttA bound to ADP is not functionally equivalent to EttA after ATP hydrolysis, when it presumably binds ADP. The data are consistent with the idea that under conditions of energy stress, when the ATP/ADP ratio decreases (such as in the stationary phase), ADP-bound EttA acts as a regulator to throttle back the cell's protein-making capacity, as this is an energy-intensive process.
G. Boël et al. The ABC-F protein EttA gates ribosome entry into the translation elongation cycle.
Nat Struct Mol Biol. 2, 143-51 (2014). doi:10.1038/nsmb.2740
B. Chen et al. EttA regulates translation by binding the ribosomal E site and restricting ribosome-tRNA dynamics.
Nat Struct Mol Biol. 2, 152-9 (2014). doi:10.1038/nsmb.2741