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 site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA-binding site. Using single-molecule fluorescence resonance energy transfer, 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, in which 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.

Figure 1

Cryo-EM 3D reconstruction of EttA-EQ₂-bound PRE complex. Overview of the segmented cryo-EM map of EttA-EQ₂-bound PRE complex reconstructed from Class I. (a) Back view showing the tRNA exit site; (b) front view showing the tRNA entry site; (c) top view; (d) intersubunit interface view from the 50S side, with 50S subunit computationally removed; (e) intersubunit interface view from the 30S side, with 30S subunit computationally removed. A-tRNA and P-tRNA, short for A-site tRNA and P-site tRNA, respectively. Abbreviations of landmarks: hd, 30S subunit head; bk, 30S subunit beak; sp, 30S subunit spur; h17, helix 17 of 16S rRNA; cp, central protuberance of 50S subunit; st, stalk; H68, H69, Helix 68 and 69 of 23S rRNA, respectively; ABC, ATP-binding cassette domain; PtIM, short for P-site tRNA interaction motif, also called inter-ABC-domain linker of EttA; arm, the arm of EttA, an α-helical ABC1 domain of EttA.

Figure 2

Ribosome E-site binding assay. Filter-binding assay evaluating the influence of increasing concentration of EttA-EQ₂ on the interaction of deacylated [³²P]tRNA^Phe (0.4 µM) with the E site on 70S ribosomes (0.2 µM) after a 2 minute incubation at 4 °C in 20 mM Tris-HCl (pH 7.4), 100 mM NH₄Cl, 10 mM Mg(OAc)₂, 0.1 mM Mg-ATP. The graph shows the fraction of ribosomes retaining an E-site tRNA after 3 washes on a nitrocellulose filter with 20 mM Tris-HCl (pH7.4), 100 mM NH₄Cl, 20 mM Mg(OAc)₂, 1mM EDTA.

Figure 3

Characterization of the global conformation of EttA-EQ₂-bound ribosome. (a, b) Superimposition of the cryo-EM map of the EttA-EQ₂-bound PRE complex determined here on that of the 70S•tRNA^fMet•MF-tRNA^Phe PRE complex in MS-I^, (a) or that of the 70S•MFTI-tRNA^Ile•EF-G•GDPNP complex with puromycin in MS-II (b), as viewed from the solvent side of the 30S subunit. (c, d, e) Comparison of EttA’s binding site on 70S ribosome (c) with those of E-site tRNA (d) and EF-P (e), as viewed from the 30S subunit side of the intersubunit interface. The 30S subunit and A-site tRNA are not shown, to provide clear visualization of the factors and P-site tRNAs. The labels used here are defined in the legend for Fig. 1. (e) A map calculated from the X-ray crystal structure of the T. thermophilus 70S ribosome complex with elongation factor EF-P, displayed in the same manner and orientation as the cryo-EM structures of the E. coli ribosomal complexes in (c, d). (f) Comparison of the positions of the L1 stalk in the 70S•EttA-EQ₂ and 70S PRE complex, showing superposition of the cryo-EM maps in the boxed regions in (c) and (d). The maps are colored the same as in (a).

Figure 4

Modeling of ATP-bound EttA monomeric structure and comparison with EttA cryo-EM density map. (a, b, c) Modeling of ATP-bound EttA monomeric structure. (a) The crystallographic structure of EttA₂ (PDB ID: 4FIN) was cut in half at the inter-ABC-domain linker region (between residues 277 and 278) to generate a monomeric apo-EttA structure model. (b) Each of the subdomains of the apo-EttA model was aligned to the head-to-tail homodimer structure of ATP-bound CFTR NBD1 (PDB ID: 2PZE), then the missing links were complemented by Phyre2 search hits, yielding a monomeric ATP-bound EttA model shown in (c). Blue, mechanical coupling subdomain; tan, ATP binding core; lighter and darker colors indicate ABC1 and ABC2, respectively. Purple, inter-ABC-domain linker, named PtIM in this paper; Grey, the other half of EttA₂ crystallographic structure; green, Mg-ATP. (d, e, f) Comparison of apo-EttA model (d) and ATP-bound EttA model (f) with isolated EttA-EQ₂ cryo-EM map (e). In (d) and (f) the cryo-EM map of EttA-EQ₂ was rendered transparent.

Figure 5

MDFF-fitted EttA-EQ₂-bound PRE complex structure. (a) Top view; (b) close-up of the boxed region in (a). (c, d, e) Stepwise close-up of the boxed region in the previous panel viewing from the 50S side of the intersubunit interface. Color scheme is the same as Fig. 1. Blue and orange ribbons, ribosomal proteins of 50S and 30S subunit, respectively. Cryo-EM map is rendered as mesh contour. The starred region in (d) indicates the protrusion formed by the CpU bulge of P-site tRNA^fMet. Residues involved in possible interactions between EttA and P-site tRNA^fMet or 23S rRNA are represented as sticks in (e).

Figure 6

EttA-mediated regulation of the open L1 stalk ↔ closed L1 stalk equilibrium of a PRE^-A_fMet complex as observed using smFRET_L1-L9. (a) Cartoon diagram of the conformational equilibrium of the PRE^-A_fMet complex between the MS-I conformation harboring an open L1 stalk (ribosomal complex on left) and the MS-II conformation harboring a closed L1 stalk (ribosomal complex on right). 30S subunit, tan cartoon; 50S subunit, blue cartoon; tRNA^fMet, green ribbon,; mRNA, black curve; Cy3 FRET donor fluorophore, green circle; and Cy5 FRET acceptor fluorophore, red circle. (b, c, d) smFRET_L1-L9 experiments recorded in the presence of 2 mM ATP and in (b) the absence of EttA, (c) the presence of 6 µM EttA, or (d) the presence of 6 µM EttA-EQ₂. 1^st row: Representative Cy3 and Cy5 fluorescence intensity (Fluor. int.) vs. time trajectories. The fluorescence intensities are plotted in arbitrary units (a.u.) with the Cy3 fluorescence intensity plotted in green and the Cy5 fluorescence intensity plotted in red. 2^nd row: The corresponding E_FRET vs. time trajectories. The E_FRET at each time point was calculated using E_FRET = I_Cy5 / (I_Cy3 + I_Cy5), where I_Cy3 and I_Cy5 are emission intensities of Cy3 and Cy5, respectively, and is plotted in blue. 3^rd row: Surface contour plots of the time evolution of the population FRET. The contour plots were generated by superimposing individual E_FRET vs. time trajectories, and colored from white (lowest-populated) to red (highest-populated). n denotes the number of E_FRET vs. time trajectories used to construct each contour plot.

Figure 7

Schematic model of the influence of EttA, in the presence of ATP, on the early steps in protein synthesis on the ribosome. For simplicity, not all intermediate steps in translation are shown; see text for more details. Tan, 30S; navy, 50S; orange, EF-Tu. Abbreviations: 70S IC, 70S ribosomal initiation complex; TC, aminoacyl-tRNA•EF-Tu•GTP ternary complex. Three yellow dots in tandem represent the heading peptide excluding the two most recently added amino acids (red circles). Blue arrow indicates the propensity of PRE complexes for spontaneous ratchet-like intersubunit rotation. Red stop sign symbolizes the inhibition of the MS-I↔MS-II dynamics by the binding of the ATP-bound form of EttA to the ribosomal E site. EttA-mediated translation regulation in the presence of ADP, described in the companion paper by Boel et al, is not represented in this schematic.