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tRNA mimicry in translation termination and beyond

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Abstract The mechanism of translation termination has long been a puzzle. The release factor (RF) class of translation factors plays a key role in terminating protein synthesis. Bacteria have two RFs, RF1 and RF2, with high specificity to decipher three stop codons. Decades ago, an idea was formulated that RFs may be protein analogs of tRNA. This idea gained substantial support 10 years ago by the identification of two classes of crucial RF peptide motifs, P(A/V)T/SPF and GGQ, in bacteria. These motifs were functionally equivalent to the anticodon and aminoacyl‐CCA terminus of tRNA, although the processes these molecules function in are different. These findings reinforced the ‘molecular mimicry’ or ‘tRNA mimicry’ hypothesis. Since then, the RF‐tRNA mimicry hypothesis has played a crucial role to elucidate the mechanism of translation termination. In the past decade, the crystal structure of the translation termination complex between the ribosome and RFs has been determined at atomic resolution. Overall, the structural data strongly support the RF‐tRNA mimicry hypothesis, with shared as well as distinct ribosomal conformations induced by RF or tRNA binding. In this review, we re‐evaluate the structural data from the genetic and biochemical viewpoint as our initial functional evidence were not fully interpreted in the previous reports. Recent structural and functional studies of the translation machinery have uncovered that the concept of tRNA mimicry can be expanded for factors beyond translation termination and into translation initiation, elongation, as well as mRNA surveillance pathways for protein synthesis. WIREs RNA 2011 2 647–668 DOI: 10.1002/wrna.81 This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes Translation > Ribosome Structure/Function Translation > Translation Regulation RNA Processing > tRNA Processing

Structural resemblance between the Dom34·Hbs1 complex and the aPelota·archaeal elongation factor 1α (aEF1α) complex. Left panel, the Dom34·Hbs1 complex from Schizosaccharomyces pombe (PDB code: 3MCA).101 Right panel, the aPelota·aEF1α complex from Aeropyrum pernix (PDB code: 3AGJ).76 Figures were rendered using Pymol.25

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Structures of the ribosome associated with initiation and elongation factors, and apo‐form crystal structures of elongation factors. The ribosomal RNAs are colored gray, the 50S subunit proteins are colored green, and the 30S subunit proteins are colored dark blue. The P‐site tRNA is shown in light blue, and the E‐site tRNA is shown in yellow as surface representations. (a) The structure of elongation factor P (EF‐P) bound to the ribosome. Overview of EF‐P and P‐site tRNA–binding in the 70S ribosome (PDB code: 3HUW, 3HUX, 3HUY, and 3HUZ).85 EF‐P is shown in magenta. (b) Left, the Thermus aquaticus EF‐Tu·GDPNP·Phe‐tRNAPhe complex (Protein Data Bank accession code 1TTT) (EF‐Tu, elongation factor Tu); right, Thermus thermophilus EF‐G·GDP (PDB code: 1DAR) (EF‐G, elongation factor G). (c) Structure of EF‐Tu and aminoacyl‐tRNA bound to the ribosome.60 Overall view of the complex, with EF‐Tu and tRNAs. Note that aminoacyl‐tRNA (orange) bound to EF‐Tu (purple) is in the ‘A/T state’. (d) EF‐G in the posttranslocational state of the ribosome.86 Overall view of EF‐G (purple) on the ribosome. Figures were rendered using Pymol.25

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Structural resemblance between elongation, termination, and surveillance complexes. (a) The complex structure of aPelota·archaeal elongation factor 1α (aEF1α)·GTP (PDB code: 3AGJ).67 The switch regions I and II of aEF1α are shown as sw1 and sw2. Loop A and loop B are marked in aPelota. (b) The complex model for aRF1·aEF1α·GTP [PDB code: 3AGK (aRF1) and 3AGJ (aEF1α)].67 AP represents ATP binding pocket. (c) The complex model for tRNA·aEF1α·GTP [PDB code: 2WRN (tRNA; mRNA) and 3AGJ (aEF1α)]. AC represents anticodon. Figures were rendered using Pymol.25

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Shared topology of domain 1 of ribosome‐bound RF2 and the elongation factor Tu (EF‐Tu)·tRNA complex. Left panel, EF‐Tu (purple) complexed with aminoacyl‐tRNA (brown) in the ‘A/T state’ (pre‐state).59 Right panel, aminoacyl‐tRNA in the A site (post‐state). RF2 (red) bound to the ribosome is superimposed.38 The P‐site tRNA (blue), E‐site tRNA (yellow), mRNA (green), 23S rRNA (orange) are shown. L11 (white) with mutations (spheres) that suppress the RF defect, and RF2 mutations (spheres) suppressing the ‘harmful’ tripeptide changes are indicated. Figures were rendered using Pymol (from PDB code: 3F1E and 3F1F).25

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Interaction of RF2 with the peptidyl‐transferase center (PTC). (a) The PTC of the ribosome showing the GGQ motif of RF2 and deacylated P‐site tRNA. The terminal CCA of P‐site tRNA (blue), the conserved GGQ motif of RF2 (red), and key bases of 23S RNA (green) are indicated.31 The main‐chain amide of Gln‐253 is positioned within hydrogen bonding distance of the 3′OH of A76 of the deacylated tRNA bound in the P site (yellow dotted line). (b) Modeling of the reaction mechanism for translation termination.31 Figures were rendered using Pymol (from PDB code: 2WH1).25

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Spatial localization of suppressor mutations in RF2 that rescue growth inhibition caused by variant RF2 proteins containing ‘harmful’ tripeptide‐anticodon changes.42,43 These ‘harmful’ changes include a Phe‐to‐Thr substitution in the SPF motif, and are indicated as ‘DL (dominant lethal) mutations’ in blue. The isolated suppressors (designated Sups) that contained charge‐flip substitutions for Glu residues are shown as magenta spheres in the RF2·ribosome complex structure.40 P represents the P‐site tRNA, and stop codon bases are shown in green. Rotated views (180°) are shown (from PDB code: 2WH1). Figures were rendered using Pymol.25

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Assignment of release factor (RF) peptides essential for discriminating stop codons by genetics.20,41 (a) Active RF hybrids. Seven conserved segments (A through G) were swapped between bacterial RF1 and RF2 sequences. The activity of the hybrid proteins was monitored by in vivo complementation tests for RF1 null and RF2 null strains as well as by the in vitro fMet release assay. A pair of RF hybrids, ΨRF1 and ΨRF2, exerted RF1‐ and RF2‐specific complementation activities due to insertion of their respective D segments. A hybrid RF construct, designated ΨRF, which is identical to ΨRF1 and ΨRF2 except for domain D, was used as a test RF backbone to monitor the specificity of transplanted peptides, and the amino acid swaps made to identify the discriminator peptide (shown as ‘PA’).20 Blue and red boxes represent RF1 and RF2 segments, respectively. Discriminator‐peptide swapping was conducted within the segment D sub‐region shown in (c). (b and c) Shuffling of conserved amino acids between RF1 and RF2 sequences at the assigned discriminator region. The indicated five residues are differentially conserved in RF1s and RF2s of many species. Each position was partially randomized using primer sequences: position Q/V contained Q, V, E, and L; position P/S contained P and S; position A/P contained A and P; position T/F contained T, F, I, and S; and position I/R contained I, R, L, and S. Hybrid ΨRF1 and ΨRF2 proteins containing all shuffled combinations (i.e., 256 cases) were synthesized and transformed into RF1 null and RF2 null strains. The number of selected amino acids at each position through each mutant selection and the specificity are shown in (b). Blue and red represent RF1‐ and RF2‐type activities, respectively. Interactions defined in the crystal structures31,38–40 are shown in (c) with the stop codon base position numbered in green: blue line, RF1‐specific interaction with stop codons; red line, RF2‐specific interaction with stop codons; purple dotted blue, RF1/RF2 common interaction with stop codons. Figures were rendered using Pymol (from PDB code: 3D5A).25

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Recognition of the stop codon in the decoding center (DC) by RF1 and RF2. (a) RF1 (the PVT motif) reading UAA (PDB code: 3D5A).38 (b) RF1 (the PVT motif) reading UAG (PDB code: 3MR8).39 (c) RF2 (the SPF motif) reading UAA (PDB code: 3F1E).31 (d) RF2 (the SPF motif) reading UGA (PDB code: 2WH1).40

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Conformational changes at the decoding center (DC) induced by binding to (a) cognate tRNA (from PDB code: 2WDK) and (b) RF1 (from PDB code: 3D5A). Sense (a) and stop (b) codons are shown in green, and the three conserved bases of 16S rRNA, A1492, A1493 and G530, are shown in gray. The anticodon of tRNA (a) and the codon discriminator motif (PA) of RF1 (b) are shown as spheres.

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Structures of RF1 and RF2 in the termination complex. (a) Model of Thermus thermophilus RF2 (red) bound to the ribosome with tRNA (yellow/blue) and mRNA (green) containing a UAA stop codon.31 (b) The structures of apo‐form T. thermophilus RF2 (‘closed’ form, PDB code: 1GQE) and its ribosome‐bound conformation (‘open’ form, PDB code: 3F1E). The crystal structure of the complex between Escherichia coli RF1 and PrmC bound to the methyl donor product AdoHCy is also shown (PDB code: 2B3T). Both the GGQ domain and the central region (domains 2 and 4) of RF1 interact with PrmC.32 Figures were rendered using Pymol.25

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Apo‐form crystal structures of class‐I release factors (RFs). Left upper panel: Escherichia coli RF2 (PDB code: 1GQE)23, with the codon discriminator motif shown as ‘PA’, which contains five amino acid residues including the tripeptide motif and two flanking residues Q/V and I/R as shown in Figure 6(c). Left lower panel: Thermotoga maritima RF1 (PDB code: 1RQ0).24 Right panel: human eRF1 (PDB code: 1DT9).22 The TASNIKS and YxCxxxF motifs are marked in different shades of red. The position of the GGQ motif and domain names are shown. Figures were rendered using Pymol.25

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Essential steps in translation. The 30S (blue) and 50S (green) subunits harboring the decoding center (DC) and peptidyl‐transferase center (PTC), with the mRNA (yellow) bound to the 30S subunit. The three different positions for tRNA molecules are indicated as the A, P, and E sites. In the elongation cycle, the incoming aminoacyl‐tRNA is delivered to the empty A site of the ribosome as a ternary complex with elongation factor Tu (EF‐Tu; purple) and GTP. After GTP hydrolysis, the peptidyl‐tRNA in the P site donates its growing polypeptide to the amino acid on the tRNA in the A site. The newly formed peptidyl‐tRNA is then translocated from the A to the P site. Simultaneously, the deacylated tRNA in the P site is moved into the E site. Protein synthesis terminates when a stop codon enters the DC of the ribosome. Release factor (RF; red) binds to the empty A site of the termination state of the ribosome, with a deacylated tRNA in the E‐site and a peptidyl‐tRNA in the P‐site, recognizes the stop codon, and triggers peptidyl‐tRNA hydrolysis.

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Docking model of the aPelota·archaeal elongation factor 1α (aEF1α) complex on the ribosome.67 (a) Overall view of the model. Colors are the same as in Figure 11 except for aPelota (red) and aEF1α (brown). (b) Transition from the pre‐state (A/T state) conformation to the post‐state (A/A site) conformation of the ribosome. aPelota bound to aEF1α is in the distorted ‘A/T state’ conformation; upon GTP hydrolysis, aEF1α is released, and the aPelota springs into the A site (as shown by the arrow) and places the amino acid in the peptidyl‐transferase center (PTC). Figures were rendered using Pymol.25

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RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Processing > tRNA Processing
Translation > Ribosome Structure/Function
Translation > Translation Regulation

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