Yoshikazu Nakamura

A tripeptide 'anticodon' deciphers stop codons in messenger RNA

The two translational release factors of prokaryotes, RF1 and RF2, catalyse the termination of polypeptide synthesis at UAG/UAA and UGA/UAA stop codons, respectively. However, how these polypeptide release factors read both non-identical and identical stop codons is puzzling. Here we describe the basis of this recognition. Swaps of each of the conserved domains between RF1 and RF2 in an RF1–RF2 hybrid led to the identification of a domain that could switch recognition specificity. A genetic selection among clones encoding random variants of this domain showed that the tripeptides Pro-Ala-Thr and Ser-Pro-Phe determine release-factor specificity in vivo in RF1 and RF2, respectively. An in vitro release study of tripeptide variants indicated that the first and third amino acids independently discriminate the second and third purine bases, respectively. Analysis with stop codons containing base analogues indicated that the C2 amino group of purine may be the primary target of discrimination of G from A. These findings show that the discriminator tripeptide of bacterial release factors is functionally equivalent to that of the anticodon of transfer RNA, irrespective of the difference between protein and RNA.

Functional sites of interaction between release factor RF1 and the ribosome.

Translational release factors decipher stop codons in mRNA and activate hydrolysis of peptidyl-tRNA in the ribosome during translation termination. The mechanisms of these fundamental processes are unknown. Here we have mapped the interaction of bacterial release factor RF1 with the ribosome by directed hydroxyl radical probing. These experiments identified conserved domains of RF1 that interact with the decoding site of the 30S ribosomal subunit and the peptidyl transferase site of the 50S ribosomal subunit. RF1 interacts with a binding pocket formed between the ribosomal subunits that is also the interaction surface of elongation factor EF-G and aminoacyl-tRNA bound to the A site. These results provide a basis for understanding the mechanism of stop codon recognition coupled to hydrolysis of peptidyl-tRNA, mediated by a protein release factor.

Emerging Understanding of Translation Termination

Most textbooks end the description of protein synthesis with the RF-mediated release of the completed polypeptide chain from the peptidyl tRNA. This is a gross oversight because there is an additional crucial step in protein synthesis: recycling of ribosomes through decomposition of the termination complex (Hirashima and Kaji 1970xHirashima, A. and Kaji, A. Biochem. Biophys. Res. Communs. 1970; 41: 877–883Crossref | PubMed | Scopus (33)See all ReferencesHirashima and Kaji 1970). In bacteria, this process requires a ribosome recycling factor (RRF, originally called ribosome releasing factor; Janosi et al. 1994xJanosi, L., Shimizu, I., and Kaji, A. Proc. Natl. Acad. Sci. USA. 1994; 91: 4249–4253Crossref | PubMedSee all ReferencesJanosi et al. 1994). This process is fundamental because the gene for RRF is essential for cell growth and the living cell must reuse the ribosome, RF, and tRNA for the next round of protein synthesis.Where does RRF bind and how does it work? Upon release of the polypeptide chain, the ribosomal A site remains occupied with a tRNA-mimicking RF protein. A translocase is probably required to forward deacylated tRNA and RF to the E and P sites of the ribosome, respectively. We speculate that RF-3 or EF-G may catalyze this final translocation reaction. RRF is known to act in vitro on a complex of mRNA with ribosomes having an empty A site and bound deacylated tRNA at the P site (Hirashima and Kaji 1970xHirashima, A. and Kaji, A. Biochem. Biophys. Res. Communs. 1970; 41: 877–883Crossref | PubMed | Scopus (33)See all ReferencesHirashima and Kaji 1970). Therefore, it is tempting to speculate that RRF also has a tRNA-mimicry domain for binding to the A site of the ribosome. The mechanism of decomposition of the termination complex by RRF and the eukaryotic RRF proteins remains to be investigated.

Crystal structure and functional analysis of the eukaryotic class II release factor eRF3 from S. pombe

Translation termination in eukaryotes is governed by two interacting release factors, eRF1 and eRF3. The crystal structure of the eEF1alpha-like region of eRF3 from S. pombe determined in three states (free protein, GDP-, and GTP-bound forms) reveals an overall structure that is similar to EF-Tu, although with quite different domain arrangements. In contrast to EF-Tu, GDP/GTP binding to eRF3c does not induce dramatic conformational changes, and Mg(2+) is not required for GDP binding to eRF3c. Mg(2+) at higher concentration accelerates GDP release, suggesting a novel mechanism for nucleotide exchange on eRF3 from that of other GTPases. Mapping sequence conservation onto the molecular surface, combined with mutagenesis analysis, identified the eRF1 binding region, and revealed an essential function for the C terminus of eRF3. The N-terminal extension, rich in acidic amino acids, blocks the proposed eRF1 binding site, potentially regulating eRF1 binding to eRF3 in a competitive manner.

The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast.

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.

Making sense of mimic in translation termination.

The mechanism of translation termination has long been a puzzle. Recent crystallographic evidence suggests that the eukaryotic release factor (eRF1), the bacterial release factor (RF2) and the ribosome recycling factor (RRF) all mimic a tRNA structure, whereas biochemical and genetic evidence supports the idea of a tripeptide anticodon in bacterial release factors RF1 and RF2. However, the suggested structural mimicry of RF2 is not in agreement with the tripeptide anticodon hypothesis and, furthermore, recently determined structures using cryo-electron microscopy show that, when bound to the ribosome, RF2 has a conformation that is distinct from the RF2 crystal structure. In addition, hydroxyl-radical probings of RRF on the ribosome are not in agreement with the simple idea that RRF mimics tRNA in the ribosome A-site. All of this evidence seriously questions the simple concept of structural mimicry between proteins and RNA and, thus, leaves only functional mimicry of protein factors of translation to be investigated.

Structural insights into eRF3 and stop codon recognition by eRF1.

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1s M domain contacts eRF3s GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1s stimulatory effect on eRF3s GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1s putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.

Yeast [PSI+] “Prions” that Are Crosstransmissible and Susceptible beyond a Species Barrier through a Quasi-Prion State

The yeast [PSI(+)] element represents an aggregated form of release factor Sup35p and is inherited by a prion mechanism. A species barrier prevents crosstransmission of the [PSI(+)] state between heterotypic Sup35p prions. Kluyveromyces lactis and Yarrowia lipolytica Sup35 proteins, however, show interspecies [PSI(+)] transmissibility and susceptibility and a high spontaneous propagation rate. Cross-seeding was visualized by coaggregation of differential fluorescence probes fused to heterotypic Sup35 proteins. This coaggregation state, referred to as a quasi-prion state, can be stably maintained as a heritable [PSI(+)] element composed of heterologous Sup35 proteins. K. lactis Sup35p was capable of forming [PSI(+)] elements not only in S. cerevisiae but in K. lactis. These two Sup35 proteins contain unique multiple imperfect oligopeptide repeats responsible for crosstransmission and high spontaneous propagation of novel [PSI(+)] elements.

The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination

The ribosomal peptidyl transferase center is responsible for two fundamental reactions, peptide bond formation and nascent peptide release, during the elongation and termination phases of protein synthesis, respectively. We used in vitro genetics to investigate the functional importance of conserved 23S rRNA nucleotides located in the peptidyl transferase active site for transpeptidation and peptidyl-tRNA hydrolysis. While mutations at A2451, U2585, and C2063 (E. coli numbering) did not significantly affect either of the reactions, substitution of A2602 with C or its deletion abolished the ribosome ability to promote peptide release but had little effect on transpeptidation. This indicates that the mechanism of peptide release is distinct from that of peptide bond formation, with A2602 playing a critical role in peptide release during translation termination.

Inhibition of midkine alleviates experimental autoimmune encephalomyelitis through the expansion of regulatory T cell population

CD4+CD25+ regulatory T (Treg) cells are crucial mediators of autoimmune tolerance. The factors that regulate Treg cells, however, are largely unknown. Here, we show that deficiency in midkine (MK), a heparin-binding growth factor involved in oncogenesis, inflammation, and tissue repair, attenuated experimental autoimmune encephalomyelitis (EAE) because of an expansion of the Treg cell population in peripheral lymph nodes and decreased numbers of autoreactive T-helper type 1 (TH1) and TH17 cells. MK decreased the Treg cell population ex vivo in a dose-dependent manner by suppression of STAT5 phosphorylation that is essential for Foxp3 expression. Moreover, administration of anti-MK RNA aptamers significantly expanded the Treg cell population and alleviated EAE symptoms. These observations indicate that MK serves as a critical suppressor of Treg cell expansion, and inhibition of MK using RNA aptamers may provide an effective therapeutic strategy against autoimmune diseases, including multiple sclerosis.