Structure of a human cap-dependent 48S translation pre-initiation complex.

Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition.

Dual function of UPF3B in early and late translation termination.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance pathway that recognizes and degrades mRNAs with premature termination codons (PTCs). The mechanisms underlying translation termination are key to the understanding of RNA surveillance mechanisms such as NMD and crucial for the development of therapeutic strategies for NMD-related diseases. Here, we have used a fully reconstituted in vitro translation system to probe the NMD proteins for interaction with the termination apparatus. We discovered that UPF3B (i) interacts with the release factors, (ii) delays translation termination and (iii) dissociates post-termination ribosomal complexes that are devoid of the nascent peptide. Furthermore, we identified UPF1 and ribosomes as new interaction partners of UPF3B. These previously unknown functions of UPF3B during the early and late phases of translation termination suggest that UPF3B is involved in the crosstalk between the NMD machinery and the PTC-bound ribosome, a central mechanistic step of RNA surveillance.

PABP enhances release factor recruitment and stop codon recognition during translation termination.

Poly(A)-binding protein (PABP) is a major component of the messenger RNA-protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro translation system, we show that PABP directly stimulates translation termination. PABP increases the efficiency of translation termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in translation termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-termination complexes (preTCs) significantly increases the efficiency of peptidyl-tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1-eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in translation termination.

Backbone (1)H, (13)C and (15)N resonance assignments of the human eukaryotic release factor eRF1.

Eukaryotic translation termination is mediated by two interacting release factors, eukaryotic class 1 release factor (eRF1) and eukaryotic class 3 release factor (eRF3), which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. eRF1 consisting of three well-defined functional domains recognizes all three mRNA stop codons located in the A site of the small ribosomal subunit and triggers hydrolysis of the ester bond of peptidyl-tRNA in the peptidyl transfer center of the large ribosomal subunit. Nevertheless, various aspects of molecular mechanism of translation termination in eukaryotes remain unclear. Elucidation of the structure and dynamics of eRF1 in solution is essential for understanding molecular mechanism of its function in translation termination. To approach this problem, here we report NMR backbone signal assignments of the human eRF1 (437 a.a., 50 kDa).

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.

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 ß-core of the protein. Such structural plasticity may be essential for stop codon recognition by human eRF1.

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.

Translation termination in pyrrolysine-utilizing archaea.

Although some data link archaeal and eukaryotic translation, the overall mechanism of protein synthesis in archaea remains largely obscure. Both archaeal (aRF1) and eukaryotic (eRF1) single release factors recognize all three stop codons. The archaeal genus Methanosarcinaceae contains two aRF1 homologs, and also uses the UAG stop to encode the 22nd amino acid, pyrrolysine. Here we provide an analysis of the last stage of archaeal translation in pyrrolysine-utilizing species. We demonstrated that only one of two Methanosarcina barkeri aRF1 homologs possesses activity and recognizes all three stop codons. The second aRF1 homolog may have another unknown function. The mechanism of pyrrolysine incorporation in the Methanosarcinaceae is discussed.

The crucial role of conserved intermolecular H-bonds inaccessible to the solvent in formation and stabilization of the TL5.5 SrRNA complex.

Analysis of the structures of two complexes of 5 S rRNA with homologous ribosomal proteins, Escherichia coli L25 and Thermus thermophilus TL5, revealed that amino acid residues interacting with RNA can be divided into two different groups. The first group consists of non-conserved residues, which form intermolecular hydrogen bonds accessible to solvent. The second group, comprised of strongly conserved residues, form intermolecular hydrogen bonds that are shielded from solvent. Site-directed mutagenesis was used to introduce mutations into the RNA-binding site of protein TL5. We found that replacement of residues of the first group does not influence the stability of the TL5.5 S rRNA complex, whereas replacement of residues of the second group leads to destabilization or disruption of the complex. Stereochemical analysis shows that the replacements of residues of the second group always create complexes with uncompensated losses of intermolecular hydrogen bonds. We suggest that these shielded intermolecular hydrogen bonds are responsible for the recognition between the protein and RNA.