Human Protein Synthesis Requires aminoacyl-tRNA Pivoting During Proofreading

Abstract

Rigorous studies have characterized the aa-tRNA selection mechanism in bacteria, which is essential for maintaining translational fidelity. Recent investigations have identified critical distinctions in humans, such as the requirement of subunit rolling and a tenfold slower proofreading step. Although these studies captured key intermediates involved in tRNA selection, they did not elucidate the transitions of aa-tRNA between intermediates. Here, we simulated 1,856 aa-tRNA accommodation events into the human ribosomal A site, revealing the requirement of a distinct ~30° pivoting of aa-tRNA about the anticodon stem within the accommodation corridor. This pivoting is crucial for navigating the crowded accommodation corridor, which becomes more constrained due to subunit rolling. Subunit rolling-dependent crowding increases the steric contributions of the accommodation corridor during aa-tRNA accommodation, consistent with the 10-fold reduction in the rate of proofreading. The pivoting of the aa-tRNA enables precise alignment within the accommodation corridor, allowing it to traverse the narrower passage. Furthermore, we found that domain III of eEF1A interacts with the accommodating aa-tRNA through conserved basic residues, providing a steric block to prevent dissociation from the A site. Together, these findings provide a structural framework for understanding the distinctions between bacterial and human aa-tRNA selection and demonstrate that the alignment of the aa-tRNA relative to the ribosomal catalytic sites is a critical determinant of translational fidelity.

Figure 1.. Conformations of ribosomes during eukaryotic aa-tRNA accommodation.

(A) All-atom structural representation of the pre-accommodated GTPase activated state with aa-tRNA in the A/T position. (B) Simplified model of the GTPase activated state with aa-tRNA in the A/T position. (C) All-atom structural representation of the accommodated state with aa-tRNA in the A/A position. (D) Simplified model of the accommodated state with aa-tRNA in the A/A position. In these models aa-tRNA (yellow), mRNA (green), peptidyl-tRNA (orange), 28S, 5S, 5.8S rRNA (white), 18S (grey), ribosomal proteins (blue) and eEF1A (purple) are represented.

Figure 2.. Dynamics of human aa-tRNA accommodation.

(A) Distance measured between O3’ atoms of U60 and U8 of the aa-tRNA and peptidyl-tRNA, respectively, to characterize the elbow accommodation dynamics of the aa-tRNA (${\mathsf{R}}_{\mathsf{elbow}}$). (B) Distance measurement between the O3’ atoms of A76 of the aa-tRNA and peptidyl tRNA during accommodation into the A site of the ribosome $({\mathsf{R}}_{\mathsf{CCA}})$. (C) Representative distance of ${\mathsf{R}}_{\mathsf{elbow}}$ during simulation using potential 2. (D) Representative distance of ${\mathsf{R}}_{\mathsf{CCA}}$ during a simulation using potential 2. (E) Representative distance of ${\mathsf{R}}_{\mathsf{elbow}}$ during a simulation using potential 1, showing reversible fluctuations. (F) Representative distance of ${\mathsf{R}}_{\mathsf{CCA}}$ during a simulation using potential 1, showing reversible fluctuations.

Figure 3.. Human aa-tRNA accommodation occurs through a 3’-CCA end dependent pathway.

(A) Approximate free energy $(\Delta {\mathsf{G}}^{\ast })$ landscape of tRNA accommodation as measured by comparison of the ${\mathsf{R}}_{\mathsf{elbow}}$ and ${\mathsf{R}}_{\mathsf{CCA}}$ distance measurements from simulations using potential 1. (B) Representative structure of EA-1. Complete 80S structure of EA-1 (i). Representation of the accommodation corridor of the ribosome with aa-tRNA engaging H89 in EA-1 (ii). Zoom in of accommodation corridor with aa-tRNA engaging H89 in EA-1 (iii). Representation of the accommodation corridor in EA-1 from the LSU perspective (iv). Zoom in of the 3’ CCA end of the aa-tRNA interacting with H71 in EA-1 (v). (C) Representative structure of the accommodation corridor during transition path from EA-1 to EA-2 (Path). Complete 80S structure of Path (i). Representation of the accommodation corridor of the ribosome with aa-tRNA engaging H71 in Path (ii). Zoom in of the Path position with the aa-tRNA interacting with H89 (iii). Representation of the accommodation corridor in Path from the LSU perspective (iv). Zoom in of the 3’ CCA end of the aa-tRNA interacting with H71 in Path (v). (D) Representative structure of EA-2. Complete 80S structure of EA-2 (1). Representation of the accommodation corridor of the ribosome wiith aa-tRNA engaging H71 after passing H89 (ii). Zoom in of the EA-2 position with the aa-tRNA interacting with H89 (iii).Representation of the accommodation corridor in EA-2 from the LSU perspective (iv). Zoom in of the 3’ CCA end of the aa-tRNA interacting with H71 in EA-2(v). In these models ${\mathsf{R}}_{\mathsf{elbow}}$ highlighted in blue and ${\mathsf{R}}_{\mathsf{CCA}}$ highlighted in red, while the rRNA (white and grey), ribosomal proteins (blue), peptidyl-tRNA (orange), aa-tRNA (yellow), H89 (grey), H90 (mauve), H71 (green), h18 (purple), H44 (red), and mRNA (green) are represented.

Figure 4.. Human aa-tRNA pivoting during accommodation into the ribosomal A site.

(A) Structural representation of aa-tRNA pivoting (${\theta }_{\mathsf{tRNA}}$) during accommodation into the A site. Perspective from the central protuberance (left), LSU (middle), and P site (right) are represented to highlight the pivoting of the aa-tRNA. Peptidyl-tRNA (orange), mRNA (green), pre-pivot aa-tRNA (yellow), and post-pivot aa-tRNA (blue) are represented and the axis of the pivoting along the anticoding stem to the elbow domain is highlighted in red. ${\theta }_{\mathsf{tRNA}}$ is the angle of the tRNA as it moves into the ribosomal A site as measured by changes in the vector perpendicular to the plane of the tRNA (Methods). (B) Cartoon representations of the aa-tRNA pivoting into the ribosomal A site. aa-tRNA begins accommodation into the ribosome in the bent non-pivoted position (left), the tRNA then needs to pivot and accommodate through a 3’CCA-end first mechanism (middle), then the tRNA returns to a non-pivoted position once the elbow of the tRNA enters the A site. (C) Approximate free energy landscape of human tRNA accommodation into the ribosomal A site generated by comparing the ${\mathsf{R}}_{\mathsf{elbow}}$ distance and ${\theta }_{\mathsf{tRNA}}$ angle, generated from potential 1 simulations. (D) Approximate free energy landscape of human tRNA accommodation into the ribosomal A site generated by comparing the ${\mathsf{R}}_{\mathsf{CCA}}$ distance and ${\theta }_{\mathsf{tRNA}}$ angle, generated from potential 1 simulations. (E) Representative structure of the open A site of the ribosome in the GA complex, prior to accommodation (F) Representative structure of the closed A site of the ribosome in the AC complex, after accommodation. (G) Approximate free energy landscape of human ribosome rolling during tRNA accommodation generated by comparing ${\mathsf{R}}_{\mathsf{elbow}}$ and ${\mathsf{R}}_{\mathsf{rolling}}$, generated from potential 2 simulations. (H) Approximate free energy landscape of human ribosome rolling during tRNA accommodation generated by comparing ${\mathsf{R}}_{\mathsf{CCA}}$ and ${\mathsf{R}}_{\mathsf{rolling}}$, generated from potential 2 simulations. (I) Histogram of distances between atoms A35 and C61 of the accommodating H. sapiens and E. coli aa-tRNA, representing the compression of tRNA during accommodation (${\mathsf{R}}_{\text{compression}}$), generated from potential 2 simulations. (J) Solvent accessible surface area (SASA) of aa-tRNA during H. sapiens and E. coli during accommodation, generated from potential 2 simulations.

Figure 5. aa-tRNA interactions with the accommodation corridor.

(A) Regions of the 28S rRNA that are within 4 Å of the aa-tRNA during accommodation simulations of the tRNA into the ribosomal A site. (B) Regions of the 18S rRNA that are within 4 Å of the aa-tRNA during accommodation simulations of the tRNA into the ribosomal A site. (C) Structural representation of rRNA and ribosomal protein uL14 that are in proximity to aa-tRNA during accommodation. (D) Histogram of ${\theta }_{\mathsf{tRNA}}$ during accommodation of tRNA, captured from N ≥ 5 potential 2 simulations composed of full ribosome or with no H69/71 or no H89.

Figure 6. eEF1A interacts with accommodating aa-tRNA through R69 and conserved basic amino acids in Domain III.

(A) R69 of switch I of eEF1A interacts with the minor groove of the aa-tRNA adjacent to the 3’CCA end. R69 is highlighted interacting with the phosphodiester backbone of the tRNA. (B) Approximate free energy landscape of ${\mathsf{R}}_{\mathsf{elbow}}$ in comparison to the distance between eEF1A R69 and A425 of domain III $({\mathsf{R}}_{\mathsf{swi}\text{-}\mathsf{DIII}})$, generated from potential 2 simulations. This landscape indicates that for aa-tRNA to accommodate, R69 of switch I needs to pass through the 3’CCA-minor groove of the aa-tRNA and dock on domain III. (C) Approximate free energy landscape of ${\mathsf{R}}_{\mathsf{swi}\text{-}\mathsf{DIII}}$ with respect to the distance of R69 and A76 of aa-tRNA, generated from potential 2 simulations. This landscape highlights that only the path where R69 passes by the 3’CCA-minor groove is available. (D) Interactions of eEF1A domain III with in proximity to the accommodating aa-tRNA. (E) Zoomed in image of domain III of eEF1A interacting with the accommodating aa-tRNA. Basic amino acids are highlighted that could interact with the aa-tRNA. (F) Approximate free energy landscape of ${\mathsf{R}}_{\mathsf{elbow}}$ distance with respect to K378 to U55 O3’ distance, generated from potential 2 simulations. This demonstrates that domain III approaches the accommodating aa-tRNA in all simulations at an ${\mathsf{R}}_{\mathsf{elbow}}$ of ~52 Å.

Figure 7.. Model of aa-tRNA pivoting into the ribosomal A-site.

(A) Crowded environment of the human accommodation corridor enables aa-tRNA accommodation to be 10-fold slower. Additional eEF1A stays in complex with the ribosome long enough form transient interactions between domain III of eEF1A and the elbow domain of the accommodating aa-tRNA (B) aa-tRNA in human approaches the accommodation corridor until it reaches the H89 steric barrier. It then requires pivoting of the aa-tRNA to properly align with all of the elements of the accommodation corridor to enter the A site.