Stop Codon Recognition Research Articles

Evolution of the translation termination factors

During of protein synthesis, or translation, four stages are usually recognized: initiation, elongation, termination, and recycling. Translation termination involves two protein types, the factors of termination of the first class participate in recognition of stop-codons and the termination factors of the second class are GTP-ases, which stimulate activity of the first class factors. Bacteria have two proteins of class 1, RF1 and RF2 (release factor), with overlapping codon specificity; both factors are capable to recognize the codon UAA, while the codons UAG and UGA are only decoded by RF1 and RF2, respectively. In addition, bacteria contain one factor of class 2, RF3, which not only stimulates activity of RF1 and RF2, but also promotes release of the first class factors after completion of termination. In contrast to prokaryotes, eukaryotic organisms have only one termination factor of class 1, eRF1. This protein recognizes each of the three stop-codons, which results in hydrolysis of peptidyl-tRNA. Eukaryotic cells also have only one factor of class 2, eRF3.

Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells

Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined “initiation-specific” binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1Δ as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of release factor eRF1-1 from Arabidopsis thaliana.

Peptide release factor 1 (RF1) regulates the termination of translation in protein synthesis by recognizing the stop codons. The eukaryotic RF1s (eRF1s) from Arabidopsis thaliana and human have different stop-codon preferences even though they share high sequence similarity. Based on known RF1 structures, it has been suggested that the specificity depends on both the local structure and the domain arrangement, but the lack of a structure of Arabidopsis eRF1 hinders a detailed comparison. To reveal the mechanism of stop-codon recognition and compare it with that of human eRF1, one of the three Arabidopsis eRF1s, AteRF1-1, was studied and a preliminary X-ray crystallographic analysis is reported here. The protein was overexpressed in Escherichia coli and crystallized at room temperature using the vapour-diffusion method. Crystals were grown from 1.6 M lithium sulfate, 0.1 M Tris-HCl pH 8.0, 2%(v/v) PEG 400 and diffracted to 3.77 Å resolution. The data were processed in point group 622, with unit-cell parameters a = b = 136.6, c = 325.7 Å.

Interactions between peptidyl tRNA hydrolase homologs and the ribosomal release factor Mrf1 in S. pombe mitochondria

Mitochondrial translation synthesizes key subunits of the respiratory complexes. In Schizosaccharomyces pombe, strains lacking Mrf1, the mitochondrial stop codon recognition factor, are viable, suggesting that other factors can play a role in translation termination. S. pombe contains four predicted peptidyl tRNA hydrolases, two of which (Pth3 and Pth4), have a GGQ motif that is conserved in class I release factors. We show that high dosage of Pth4 can compensate for the absence of Mrf1 and loss of Pth4 exacerbates the lack of Mrf1. Also Pth4 is a component of the mitochondrial ribosome, suggesting that it could help recycling stalled ribosomes.

Identification of amino acids responsible for stop codon recognition for polypeptide chain release factor

One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.

A Single Amino Acid Substitution Alters Omnipotent eRF1 of Dileptus to Euplotes-type Dualpotent eRF1: Standard Codon Usage May be Advantageous in Raptorial Ciliates

Most ciliates use a deviant genetic code. Eukaryotic release factor (eRF1) appears to play an important role in the process of reassignment of stop codons. Although the precise site on eRF1 for recognition of stop codons remains obscure, studies have suggested that the tip region NIKS and its adjacent YxCxxxF motifs in domain 1 are important for stop codon recognition. Litostomatea is a class of ciliate that appears to use the standard genetic code. We used Dileptus (Litostomatea) eRF1 in this study to identify key residues located in or near the YxCxxxF motif. We predicted sites involving stop codon recognition by computational calculation of RNA-protein interaction propensity. We introduced mutations at the predicted sites of Dileptus eRF1 and examined the activity of the mutated Dileptus eRF1 using in vivo assay systems. The results show that the single mutation R128I (Dileptus eRF1 numbering) in the YxCxxxF motif converted the omnipotent recognition of Dileptus eRF1 to Euplotes-type dualpotent eRF1. Our results indicate that R128 is one of the key residues preserving the ability to recognize all three stop codons, especially UGA, in Dileptus. We discuss a possible advantage that ciliates from the Litostomatea class may gain from using the standard genetic code.

Two-step model of stop codon recognition by eukaryotic release factor eRF1

Release factor eRF1 plays a key role in the termination of protein synthesis in eukaryotes. The eRF1 consists of three domains (N, M and C) that perform unique roles in termination. Previous studies of eRF1 point mutants and standard/variant code eRF1 chimeras unequivocally demonstrated a direct involvement of the highly conserved N-domain motifs (NIKS, YxCxxxF and GTx) in stop codon recognition. In the current study, we extend this work by investigating the role of the 41 invariant and conserved N-domain residues in stop codon decoding by human eRF1. Using a combination of the conservative and non-conservative amino acid substitutions, we measured the functional activity of >80 mutant eRF1s in an in vitro reconstituted eukaryotic translation system and selected 15 amino acid residues essential for recognition of different stop codon nucleotides. Furthermore, toe-print analyses provide evidence of a conformational rearrangement of ribosomal complexes that occurs during binding of eRF1 to messenger RNA and reflects stop codon decoding activity of eRF1. Based on our experimental data and molecular modelling of the N-domain at the ribosomal A site, we propose a two-step model of stop codon decoding in the eukaryotic ribosome.

A Role for the 2′ OH of Peptidyl-tRNA Substrate in Peptide Release on the Ribosome Revealed through RF-Mediated Rescue

The 2' OH of the peptidyl-tRNA substrate is thought to be important for catalysis of both peptide bond formation and peptide release in the ribosomal active site. The release reaction also specifically depends on a release factor protein (RF) to hydrolyze the ester linkage of the peptidyl-tRNA upon recognition of stop codons in the A site. Here, we demonstrate that certain amino acid substitutions (in particular those containing hydroxyl or thiol groups) in the conserved GGQ glutamine of release factor RF1 can rescue defects in the release reaction associated with peptidyl-tRNA substrates lacking a 2' OH. We explored this rescue effect through biochemical and computational approaches that support a model where the 2' OH of the P-site substrate is critical for orienting the nucleophile in a hydrogen-bonding network productive for catalysis.

Structural basis for translation termination by archaeal RF1 and GTP-bound EF1α complex

When a stop codon appears at the ribosomal A site, the class I and II release factors (RFs) terminate translation. In eukaryotes and archaea, the class I and II RFs form a heterodimeric complex, and complete the overall translation termination process in a GTP-dependent manner. However, the structural mechanism of the translation termination by the class I and II RF complex remains unresolved. In archaea, archaeal elongation factor 1 alpha (aEF1α), a carrier GTPase for tRNA, acts as a class II RF by forming a heterodimeric complex with archaeal RF1 (aRF1). We report the crystal structure of the aRF1·aEF1α complex, the first active class I and II RF complex. This structure remarkably resembles the tRNA·EF–Tu complex, suggesting that aRF1 is efficiently delivered to the ribosomal A site, by mimicking tRNA. It provides insights into the mechanism that couples GTP hydrolysis by the class II RF to stop codon recognition and peptidyl-tRNA hydrolysis by the class I RF. We discuss the different mechanisms by which aEF1α recognizes aRF1 and aPelota, another aRF1-related protein and molecular evolution of the three functions of aEF1α.

Identification of eRF1 residues that play critical and complementary roles in stop codon recognition.

The initiation and elongation stages of translation are directed by codon-anticodon interactions. In contrast, a release factor protein mediates stop codon recognition prior to polypeptide chain release. Previous studies have identified specific regions of eukaryotic release factor one (eRF1) that are important for decoding each stop codon. The cavity model for eukaryotic stop codon recognition suggests that three binding pockets/cavities located on the surface of eRF1's domain one are key elements in stop codon recognition. Thus, the model predicts that amino acid changes in or near these cavities should influence termination in a stop codon-dependent manner. Previous studies have suggested that the TASNIKS and YCF motifs within eRF1 domain one play important roles in stop codon recognition. These motifs are highly conserved in standard code organisms that use UAA, UAG, and UGA as stop codons, but are more divergent in variant code organisms that have reassigned a subset of stop codons to sense codons. In the current study, we separately introduced TASNIKS and YCF motifs from six variant code organisms into eRF1 of Saccharomyces cerevisiae to determine their effect on stop codon recognition in vivo. We also examined the consequences of additional changes at residues located between the TASNIKS and YCF motifs. Overall, our results indicate that changes near cavities two and three frequently mediated significant effects on stop codon selectivity. In particular, changes in the YCF motif, rather than the TASNIKS motif, correlated most consistently with variant code stop codon selectivity.

Structure and dynamics in solution of the stop codon decoding N‐terminal domain of the human polypeptide chain release factor eRF1

The high-resolution NMR structure of the N-domain of human eRF1, responsible for stop codon recognition, has been determined in solution. The overall fold of the protein is the same as that found in the crystal structure. However, the structures of several loops, including those participating in stop codon decoding, are different. Analysis of the NMR relaxation data reveals that most of the regions with the highest structural discrepancy between the solution and solid states undergo internal motions on the ps-ns and ms time scales. The NMR data show that the N-domain of human eRF1 exists in two conformational states. The distribution of the residues having the largest chemical shift differences between the two forms indicates that helices α2 and α3, with the NIKS loop between them, can switch their orientation relative to the β-core of the protein. Such structural plasticity may be essential for stop codon recognition by human eRF1.

Post‐Termination Events in Eukaryotic Translation

Termination occurs when a stop codon enters the ribosomal A‐site. It consists of stop codon recognition followed by hydrolysis of the P‐site peptidyl‐tRNA. The hydrolysis step involves nucleophilic attack of water on peptidyl‐tRNA in the peptidyl transferase center of the large ribosomal subunit. In eukaryotes, termination results from the complex functional interplay between release factors eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples stop codon recognition and peptidyl‐tRNA hydrolysis by eRF1. Recycling of post‐termination complexes (post‐TCs) is promoted by the concerted action of ABCE1, an essential member of ATP‐binding cassette family of proteins, and eRF1, which remains associated with post‐TCs after peptide release. ABCE1 and eRF1 dissociate post‐TCs into free 60S and tRNA/mRNA‐bound 40S subunits. Subsequent tRNA/mRNA release from 40S subunits can be promoted either by initiation factors 1/1A/3, or by Ligatin. In some cases post‐TCs do not undergo complete recycling, and termination is instead followed by reinitiation on the same mRNA, usually downstream of the stop codon. This mostly occurs after translation of short open reading frames. To investigate the molecular mechanisms underlying reinitiation, we investigated different fates of ribosomal post‐termination complexes depending on the presence of initiation, as well as elongation, factors, and initiator or elongator tRNAs.

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site.

Methylation of class I translation termination factors: Structural and functional aspects

During protein synthesis, release of polypeptide from the ribosome occurs when an in frame termination codon is encountered. Contrary to sense codons, which are decoded by tRNAs, stop codons present in the A-site are recognized by proteins named class I release factors, leading to the release of newly synthesized proteins. Structures of these factors bound to termination ribosomal complexes have recently been obtained, and lead to a better understanding of stop codon recognition and its coordination with peptidyl-tRNA hydrolysis in bacteria. Release factors contain a universally conserved GGQ motif which interacts with the peptidyl-transferase centre to allow peptide release. The Gln side chain from this motif is methylated, a feature conserved from bacteria to man, suggesting an important biological role. However, methylation is catalysed by completely unrelated enzymes. The function of this motif and its post-translational modification will be discussed in the context of recent structural and functional studies.

Recruitment of a Ribosomal Release Factor for Light- and Stress-Dependent Regulation of petB Transcript Stability in Arabidopsis Chloroplasts

Land plant genomes encode four functional ribosomal peptide chain release factors (Prf) of eubacterial origin, two (PrfA and PrfB homologs) for each endosymbiotic organelle. Formerly, we have shown that the Arabidopsis thaliana chloroplast-localized PrfB homolog, PrfB1, is required not only for termination of translation but also for stabilization of UGA stop codon-containing chloroplast transcripts. A previously undiscovered PrfB-like protein, PrfB3, is localized to the chloroplast stroma in a petB RNA-containing complex and found only in vascular plants. Highly conserved positions of introns unequivocally indicate that PrfB3 arose from a duplication of PrfB1. Notably, PrfB3 is lacking the two most important tripeptide motifs characteristic for all eubacterial and organellar PrfB homologs described so far: the stop codon recognition motif SPF and the catalytic center GGQ for peptidyl-tRNA hydrolysis. Complementation studies, as well as functional and molecular analyses of two allelic mutations in Arabidopsis, both of which lead to a specific deficiency of the cytochrome b₆f complex, revealed that PrfB3 is essentially required for photoautotrophic growth. Plastid transcript, polysome, and translation analyses indicate that PrfB3 has been recruited in vascular plants for light- and stress-dependent regulation of stability of 3' processed petB transcripts to adjust cytochrome b₆ levels.

Structural aspects of translation termination on the ribosome

Translation of genetic information encoded in messenger RNAs into polypeptide sequences is carried out by ribosomes in all organisms. When a full protein is synthesized, a stop codon positioned in the ribosomal A site signals termination of translation and protein release. Translation termination depends on class I release factors. Recently, atomic-resolution crystal structures were determined for bacterial 70S ribosome termination complexes bound with release factors RF1 or RF2. In combination with recent biochemical studies, the structures resolve long-standing questions about translation termination. They bring insights into the mechanisms of recognition of all three stop codons, peptidyl-tRNA hydrolysis, and coordination of stop-codon recognition with peptidyl-tRNA hydrolysis. In this review, the structural aspects of these mechanisms are discussed.

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

Histidine 197 in Release Factor 1 Is Essential for A Site Binding and Peptide Release

Class I peptide release factors 1 and 2 (RF1 and RF2, respectively) recognize the stop codons in the ribosomal decoding center and catalyze peptidyl-tRNA hydrolysis. High-fidelity stop codon recognition by these release factors is essential for accurate peptide synthesis and ribosome recycling. X-ray crystal structures of RF1 and RF2 bound to the ribosome have identified residues in the mRNA-protein interface that appear to be critical for stop codon recognition. Especially interesting is a conserved histidine in all bacterial class I release factors that forms a stacking interaction with the second base of the stop codon. Here we analyzed the functional significance of this conserved histidine (position 197 in Escherichia coli) of RF1 by point mutagenesis to alanine. Equilibrium binding studies and transient-state kinetic analysis have shown that the histidine is essential for binding with high affinity to the ribosome. Furthermore, analysis of the binding data indicates a conformational change within the RF1·ribosome complex that results in a more tightly bound state. The rate of peptidyl-tRNA hydrolysis was also reduced significantly, more than the binding data would suggest, implying a defect in the orientation of the GGQ domain without the histidine residue.

Functional characterization of polypeptide release factor 1b in the ciliate Euplotes

In higher eukaryotes, RF-I (class I release factor) [eRF1 (eukaryotic release factor 1)] is responsible for stop codon recognition and promotes nascent polypeptide release from the ribosome. Interestingly, two class I RFs, eRF1a and eRF1b, have been identified among the ciliates Euplotes, which are variant code organisms. In the present study, we analysed the comparative expression of eRF1a and eRF1b in Euplotes cells, demonstrating that the expression of eRF1b was higher than that of eRF1a. An interaction between eRF1b and eRF3 was confirmed, suggesting that an eRF1b function is facilitated by eRF3. Co-localization of both eRF1s indicated that they function in the same subcellular location in Euplotes cells. We also analysed the characteristics of stop codon discrimination by eRF1b. Like eRF1a, eRF1b recognized UAA and UAG as stop codons, but not UGA. This finding disagreed with the deduced characteristics of eRF1a/eRF1b from the classic hypothesis of 'anticodon-mimicry' proposed by Muramatsu et al. [Muramatsu, Heckmann, Kitanaka and Kuchino (2001) FEBS Lett. 488, 105-109]. Mutagenesis experiments indicated that the absolutely conserved amino acid motif 'G31T32' (numbered as for human eRF1) in eRF1b was the key to efficient stop codon recognition by eRF1b. In conclusion, these findings support and improve the 'cavity model' of stop codon discrimination by eRF1 proposed by Bertram et al. [Bertram, Bell, Ritchie, Fullerton and Stansfield (2000) RNA 6, 1236-1247] and Inagaki et al. [Inagaki, Blouin, Doolittle and Roger (2002) Nucleic Acids Res. 30, 532-544].

A single amino acid change of translation termination factor eRF1 switches between bipotent and omnipotent stop-codon specificity †

In eukaryotes a single class-1 translation termination factor eRF1 decodes the three stop codons: UAA, UAG and UGA. Some ciliates, like Euplotes, have a variant code, and here eRF1s exhibit UAR-only specificity, whereas UGA is reassigned as a sense codon. Since eukaryote eRF1 stop-codon recognition is associated with its N-terminal domain, structural features should exist in the N domain of ciliate eRF1s that restrict their stop-codon specificity. Using an in vitro reconstituted eukaryotic translation system we demonstrate here that a chimeric eRF1 composed of the N domain of Euplotes aediculatus eRF1 fused to the MC domains of human eRF1 exhibits UAR-only specificity. Functional analysis of eRF1 chimeras constructed by swapping Euplotes N domain sequences with the cognate regions from human eRF1 as well as site-directed mutagenesis of human eRF1 highlighted the crucial role of the alanine residue in position 70 of E. aediculatus eRF1 in restricting UGA decoding. Switching the UAR-only specificity of E. aediculatus eRF1 to omnipotent mode is due to a single point mutation. Furthermore, we examined the influence of eRF3 on the ability of chimeric and mutant eRF1s to induce peptide release in response to different stop codons.