Ribosome dynamics during decoding

Abstract

Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNA^Sec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation.This article is part of the themed issue 'Perspectives on the ribosome'.

Keywords: decoding; recoding; ribosome; tRNA; translation.

Figure 1.

Schematic of the EF-Tu-dependent aa-tRNA delivery to the A site. The decoding pathway comprises two subsequent selection steps, initial selection and proofreading. The sequence of steps is based on ensemble kinetics and single molecule FRET experiments. Rate constants of kinetically important steps are shown for cognate (blue) and near-cognate (red) aa-tRNAs at 20°C [,–35]. The rate constants of the two irreversible steps, GTP hydrolysis and peptide bond formation, are limited by the respective preceding steps.

Figure 2.

Mechanism of UGA recoding to Sec by SelB–Sec-tRNA^Sec as delineated by cryo-EM. Labels: pep-tRNA, peptidyl-tRNA; d, domains of SelB; dark red shading, GTPase-activated state of SelB; arrows ‘open’ and ‘close’, domain opening and closure of the 30S shoulder (sh). States correspond to structural intermediates resolved by cryo-EM; state was modelled based on the structural data [13]. The initial complex contains an mRNA with a UGA stop codon in the A site and a SECIS element exposed for SelB recruitment. The ribosome is in the classical state with a peptidyl-tRNA in the P site and a vacant A site, while the universally conserved bases A1492 and A1493 of 16S rRNA fluctuate between the flipped-in and flipped-out conformations. The recruitment state is formed upon binding of SelB–GTP–Sec-tRNA^Sec to the SECIS. The contact between SelB domain 4 and the SECIS is maintained in all subsequent steps. Sec-tRNA^Sec can spontaneously sample a broad range of different conformations. In the transient initial binding state, Sec-tRNA^Sec binds to the SRL and SelB to the shoulder domain of the 30S subunit. The shoulder domain moves apart stabilizing A1492 and A1493 in a flipped-in conformation. The distance to the UGA codon decreases as tRNA^Sec attempts to read the codon in the transient codon reading state. GTPase-activated pre-hydrolysis state. Codon recognition by tRNA^Sec induces a local closure of the decoding centre with A1492 and A1493 flipping out. The resulting global closure of the shoulder domain leads to repositioning of the tRNA and docking of SelB on the SRL. The docking leads to a codon-dependent GTPase activation of SelB (dark red shading) and GTP hydrolysis. Notably, SelB, which initially interacts with the 30S shoulder only, does not interact with the SRL until the final GTPase-activated state is reached.

Figure 3.

Conformational rearrangements of the SSU upon UGA codon recognition by Sec-tRNA^Sec (modified from [13]). (a) Local rearrangements of the decoding centre on the SSU. IC, IB, CR and GA are the initial complex prior to SelB-Sec-tRNA recruitment, initial binding, codon reading and GTPase-activated states, respectively. In the IC state, A1492 and A1493 switch between the flipped-in (in) and flipped-out (out) conformations (two-headed arrow); G530 adopts the anti-configuration (anti). (b) Global changes of the SSU. Left: 16S rRNA with landmarks indicated; h44, helix 44 of 16S rRNA. Right: Displacements of the 16S rRNA backbone phosphates in the IB, CR or GA states with respect to the IC as obtained by superposition on 16S rRNA; negative values denote domain opening and positive values domain closure of the 30S subunit.

Figure 4.

Coupling between Sec-tRNA^Sec repositioning, docking of SelB on the SRL and GTPase activation (modified from [13]). (a) Sequential docking of SelB-Sec-tRNA^Sec on the SRL. Movements of the anticodon (shown by distance denoted as R_ASL) and elbow (R_elb distance) of tRNA^Sec and of SelB His61 (R_His61 distance) are indicated. Ribosome elements interacting with tRNA^Sec are shown in mauve, with SelB in pink; sh, 30S shoulder (G357 to U368 of 16S rRNA); S12, protein S12; DC, decoding centre; H43 and H89, helices of 23S rRNA; L11, protein L11. (b) Distance changes from state to state; R_ASL (N3 of C35 in tRNA^Sec to N1 of G in UGA); R_elb (tRNA^Sec elbow, C5′ of Ψ55 in tRNA^Sec to O2′ of A2473 in H89); R_His61 (ND1 of His61 in SelB to O2′ of G2661 in SRL).

Figure 5.

The active site of EF-Tu and SelB and the mechanism of GTP hydrolysis. Close up of the GTPase centre of (a) EF-Tu (PDB: 4V5 L) and of (b) SelB (PBD: 5LZD) in the activated state (modified from [13]). (c) and (d) The mechanism of GTP hydrolysis in translational GTPases. Two universally conserved residues, an Asp in the P-loop and a His in the switch II motif contribute to proper positioning of the γ-phosphate of GTP and of the water molecule, respectively. The side chain of the catalytic His is protonated upon contact with the SRL. The role/position of the Mg ion is based on the GTPase-activated pre-hydrolysis structures of EF-G (PDB 4V90, 4JUW, REF) in (c) and SelB (PDB 5LZD [13]) in (d).

Figure 6.

Model of the aa-tRNA accommodation pathway (from [28]; copyright 2005 National Academy of Sciences USA). Stage 1 represents the hypothetic state after the dissociation of EF-Tu, but before the onset of tRNA accommodation. Stage 2 shows the movement of the aminoacyl-tRNA body (except for the 3′CCA portion of tRNA) towards the PTC. In stage 3, the aminoacyl-tRNA body is accommodated. During stage 4, the aminoacyl-tRNA 3′-CCA end is accommodated into the PTC. The schematics depict the process of accommodation.