Class-1 polypeptide chain release factors (RFs) play a key role in translation termination. Eukaryotic (eRF1) and archaeal class-1 RFs possess a highly conserved Asn-Ile-Lys-Ser (NIKS) tetrapeptide located at the N-terminal domain of human eRF1. In the three-dimensional structure, NIKS forms a loop between helices. The universal occurrence and exposed nature of this motif provoke the appearance of hypotheses postulating an essential role of this tetrapeptide in stop codon recognition and ribosome binding. To approach this problem experimentally, site-directed mutagenesis of the NIKS (positions 61–64) in human eRF1 and adjacent amino acids has been applied followed by determination of release activity and ribosome-binding capacity of mutants. Substitutions of Asn61 and Ile62 residues of the NIKS cause a decrease in the ability of eRF1 mutants to promote termination reaction in vitro, but to a different extent depending on the stop codon specificity, position, and nature of the substituting residues. This observation points to a possibility that Asn-Ile dipeptide modulates the specific recognition of the stop codons by eRF1. Some replacements at positions 60, 63, and 64 cause a negligible (if any) effect in contrast to what has been deduced from some current hypotheses predicting the structure of the termination codon recognition site in eRF1. Reduction in ribosome binding revealed for Ile62, Ser64, Arg65, and Arg68 mutants argues in favor of the essential role played by the right part of the NIKS loop in interaction with the ribosome, most probably with ribosomal RNA.

Eukaryote ribosomal translation is terminated when release factor eRF1, in a complex with eRF3, binds to one of the three stop codons. The tertiary structure and dimensions of eRF1 are similar to that of a tRNA, supporting the hypothesis that release factors may act as molecular mimics of tRNAs. To identify the yeast eRF1 stop codon recognition domain (analogous to a tRNA anticodon), a genetic screen was performed to select for mutants with disabled recognition of only one of the three stop codons. Nine out of ten mutations isolated map to conserved residues within the eRF1 N-terminal domain 1. A subset of these mutants, although wild-type for ribosome and eRF3 interaction, differ in their respective abilities to recognize each of the three stop codons, indicating codon-specific discrimination defects. Five of six of these stop codon-specific mutants define yeast domain 1 residues (I32, M48, V68, L123, and H129) that locate at three pockets on the eRF1 domain 1 molecular surface into which a stop codon can be modeled. The genetic screen results and the mutant phenotypes are therefore consistent with a role for domain 1 in stop codon recognition; the topology of this eRF1 domain, together with eRF1-stop codon complex modeling further supports the proposal that this domain may represent the site of stop codon binding itself.

Translation termination is the final step that completes the synthesis of a polypeptide. Premature translation termination by introduction of a nonsense mutation leads to the synthesis of a truncated protein. We report the identification and characterization of the product of the MTT1 gene, a helicase belonging to the Upf1-like family of helicases that is involved in modulating translation termination. MTT1 is homologous to UPF1, a factor previously shown to function in both mRNA turnover and translation termination. Overexpression of MTT1 induced a nonsense suppression phenotype in a wild-type yeast strain. Nonsense suppression is apparently not due to induction of [PSI+], even though cooverexpression of HSP104 alleviated the nonsense suppression phenotype observed in cells overexpressing MTT1, suggesting a more direct role of Hsp104p in the translation termination process. The MTT1 gene product was shown to interact with translation termination factors and is localized to polysomes. Taken together, these results indicate that at least two members of a family of RNA helicases modulate translation termination efficiency in cells.

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The “core” domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.

Although the primary structures of class 1 polypeptide release factors (RF1 and RF2 in prokaryotes, eRF1 in eukaryotes) are known, the molecular basis by which they function in translational termination remains obscure. Because all class 1 RFs promote a stop-codon-dependent and ribosome-dependent hydrolysis of peptidyl-tRNAs, one may anticipate that this common function relies on a common structural motif(s). We have compared amino acid sequences of the available class 1 RFs and found a novel, common, unique, and strictly conserved GGQ motif that should be in a loop (coil) conformation as deduced by programs predicting protein secondary structure. Site-directed mutagenesis of the human eRF1 as a representative of class 1 RFs shows that substitution of both glycyl residues in this motif, G183 and G184, causes complete inactivation of the protein as a release factor toward all three stop codons, whereas two adjacent amino acid residues, G181 and R182, are functionally nonessential. Inactive human eRF1 mutants compete in release assays with wild-type eRF1 and strongly inhibit their release activity. Mutations of the glycyl residues in this motif do not affect another function, the ability of eRF1 together with the ribosome to induce GTPase activity of human eRF3, a class 2 RF. We assume that the novel highly conserved GGQ motif is implicated directly or indirectly in the activity of class 1 RFs in translation termination.

Translation termination in eukaryotes requires a codon-specific (class-I) release factor, eRF1, and a GTP/GDP-dependent (class-II) release factor, eRF3. The model of “molecular mimicry between release factors and tRNA” predicts that eRF1 mimics tRNA to read the stop codon and that eRF3 mimics elongation factor EF-Tu to bring eRF1 to the A site of the ribosome for termination of protein synthesis. In this study, we set up three systems, in vitro affinity binding, a yeast two-hybrid system, and in vitro competition assay, to determine the eRF3-binding site of eRF1 using the fission yeast Schizosaccharomyces pombe proteins and creating systematic deletions in eRF1. The in vitro affinity binding experiments demonstrated that the predicted tRNA-mimicry truncation of eRF1 (Sup45) forms a stable complex with eRF3 (Sup35). All three test systems revealed that the most critical binding site is located at the C-terminal region of eRF1, which is conserved among eukaryotic eRF1s and rich in acidic amino acids. To our surprise, however, the C-terminal deletion eRF1 seems to be sufficient for cell viability in spite of the severe defect in eRF3 binding when expressed in a temperature-sensitive sup45 mutant of the budding yeast, Saccharomyces cerevisiae. These results cannot be accounted for by the simple “eRF3–EF-Tu mimicry” model, but may provide new insight into the eRF3 function for translation termination in eukaryotes.