Mechanism of Inhibition of Translation Termination by Blasticidin S

Understanding the mechanisms of inhibitors of translation termination may inform development of new antibacterials and therapeutics for premature termination diseases. We report the crystal structure of the potent termination inhibitor blasticidin S bound to the ribosomal 70S?release factor 1 (RF1) termination complex. Blasticidin S shifts the catalytic domain 3 of RF1 and restructures the peptidyl transferase center. Universally conserved uridine 2585 in the peptidyl transferase center occludes the catalytic backbone of the GGQ motif of RF1, explaining the structural mechanism of inhibition. Rearrangement of domain 3 relative to the codon-recognition domain 2 provides insight into the dynamics of RF1 implicated in termination accuracy.     

Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis.

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.     

Bacterial polypeptide release factor RF2 is structurally distinct from eukaryotic eRF1.

Bacterial release factor RF2 promotes termination of protein synthesis, specifically recognizing stop codons UAA or UGA. The crystal structure of Escherichia coli RF2 has been determined to a resolution of 1.8 A. RF2 is structurally distinct from its eukaryotic counterpart eRF1. The tripeptide SPF motif, thought to confer RF2 stop codon specificity, and the universally conserved GGQ motif, proposed to be involved with the peptidyl transferase center, are exposed in loops only 23 A apart, and the structure suggests that stop signal recognition is more complex than generally believed.     

Mapping functionally important motifs SPF and GGQ of the decoding release factor RF2 to the Escherichia coli ribosome by hydroxyl radical footprinting. Implications for macromolecular mimicry and structural changes in RF2

The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA. For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits. In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination. In the presence of the cognate UGA stop codon, residues flanking the universally conserved (250)GGQ(252) motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers. In contrast, residues that flank the putative "peptide anticodon" of RF2, (205)SPF(207), were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center. Within the recently solved crystal structure of E. coli RF2, the GGQ and SPF motifs are separated by 23 A only, a distance that is incompatible with the observed cleavage sites that are up to 100 A apart. Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.     

The synthetase gene of the RNA phages R17, MS2 and f2 has a single UAG terminator codon.

Translation of the RNA from the wild-type bacteriophages R17, MS2, and f2 in bacterial cell-free extracts containing an amber suppressor yields 30-40% of the synthetase with an approximate molecular weight of 63 500, slightly larger than the major synthetase product (63 000 daltons). The occurrence of the 63 500 dalton in vitro product is dependent on the presence of an amber suppressor, and we predict that it is due to read-through of a UAG termination codon at the end of the synthetase gene. Previous results of Capecchi and Klein (Nature, 226, 1029-1033, 1070) showed that antibodies to both release factors RF1 and RF2 are required to block release of synthetase, suggesting that synthetase is released at a UAA codon. If the interpretations of both experiments are correct, the termination and release may not be synonomous and may be spatially separated. In addition there is the unexplained fact that 7% of the synthetase made in vitro in both su+ and su- extracts with either R17, MS2 or f2 as template has an apparent molecular weight of 66 000.     

Thermodynamic and Kinetic Insights into Stop Codon Recognition by Release Factor 1

Stop codon recognition is a crucial event during translation termination and is performed by class I release factors (RF1 and RF2 in bacterial cells). Recent crystal structures showed that stop codon recognition is achieved mainly through a network of hydrogen bonds and stacking interactions between the stop codon and conserved residues in domain II of RF1/RF2. Additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of RF1 on the ribosome, which is essential for triggering peptide release. In this study we mutated four conserved residues in Escherichia coli RF1 (Gln185, Arg186, Thr190, and Thr198) that are proposed to be critical for discriminating stop codons from sense codons. Our thermodynamic and kinetic analysis of these RF1 mutants showed that the mutations inhibited the binding of RF1 to the ribosome. However, the mutations in RF1 did not affect the rate of peptide release, showing that imperfect recognition of the stop codon does not affect the proper positioning of RF1 on the ribosome.     

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.     

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.     

Comparative study of translation termination sites and release factors (RF1 and RF2) in procaryotes

Translation termination is catalyzed by release factors that recognize stop codons. However, previous works have shown that in some bacteria, the termination process also involves bases around stop codons. Recently, Ito et al. analyzed release factors and identified the amino acids therein that recognize stop codons. However, the amino acids that recognize bases around stop codons remain unclear. To identify the candidate amino acids that recognize the bases around stop codons, we aligned the protein sequences of the release factors of various bacteria and searched for amino acids that were conserved specifically in the sequence of bacteria that seemed to regulate translation termination by bases around stop codons. As a result, species having several highly conserved residues in RF1 and RF2 showed positive correlations between their codon usage bias and conservation of the bases around the stop codons. In addition, some of the residues were located very close to the SPF motif, which deciphers stop codons. These results suggest that these conserved amino acids enable the release factors to recognize the bases around the stop codons. Present address (Y. Ozawa): Tokyo Research Laboratory, IBM Japan, Ltd., 1623-14 Shimotsuruma, Yamato-shi, Kanagawa 242-8502, Japan     

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.     

Recent advances in peptide chain termination

Peptide chain termination occurs when a stop codon is decoded by a release factor. In Escherichia coli two codon-specific release factors (RF1 and RF2) direct the termination of protein synthesis, while in eukaryotes a single factor is required. The E. coli factors have been purified and their genes isolated. A combination of protein and DNA sequence data reveal that the RFs are structurally similar and that RF2 is encoded in two reading frames. Frame-shifting from one reading frame to the next occurs at a rate of 50%, is regulated by the RF2-specific stop codon UGA, and involves the direct interaction of the RF2 mRNA with the 3' end of the 16S rRNA. The RF genes are located in two separate operons, with the RF1 gene located at 26.7 min and the RF2 gene at 62.3 min on the chromosome map. Ribosomal binding studies place the RF-binding region at the interface between the ribosomal subunits. A possible mechanism of stop-codon recognition is reviewed.     

Cloning of the Mycoplasma capricolum gene encoding peptide-chain release factor

In Mycoplasma capricolum (Mc), a relative of Gram⁺ eubacteria with a high genomic A+T-content, the UGA codon is assigned to Trp instead of being a stop codon. We previously showed the lack of peptide-chain release factor (RF) activity in vitro responding to the UGA codon in this bacterium [Inagaki et al., Nucleic Acids Res. 21 (1993) 1335–1338]. To obtain more information on the translation termination mechanism of Mc, we isolated and sequenced the gene encoding RF. The deduced amino-acid sequence has no RF-2-specific + 1 frameshift site and shows 50 and 36% identity to Escherichia coli RF-1 and RF-2, respectively. We conclude that this gene encodes the putative RF-1 which would possess the conserved ‘five-domain’ structure of RF family found in various organisms     

Two distinct components of release factor function uncovered by nucleophile partitioning analysis

During translation termination, release factor (RF) protein catalyzes a hydrolytic reaction in the large subunit peptidyl transferase center to release the finished polypeptide chain. While the mechanism of catalysis of peptide release remains obscure, important contributing factors have been identified, including conserved active-site nucleotides and a GGQ tripeptide motif in the RF. Here we describe pre-steady-state kinetic and nucleophile competition experiments to examine RF contributions to the rate and specificity of peptide release. We find that while unacylated tRNA stimulates release in a nondiscriminating manner, RF1 is very specific for water. Further analysis reveals that amino acid Q235 of the RF1 GGQ motif is critical for the observed specificity. These data lead to a model where RFs make two distinct contributions to catalysis--a relatively nonspecific activation of the catalytic center and specific selection of water as a nucleophile facilitated by Q235.     

Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.

Termination of translation in higher organisms is a GTP-dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.     

2A peptides provide distinct solutions to driving stop-carry on translational recoding

Expression of viral proteins frequently includes non-canonical decoding events ('recoding') during translation. '2A' oligopeptides drive one such event, termed 'stop-carry on' recoding. Nascent 2A peptides interact with the ribosomal exit tunnel to dictate an unusual stop codon-independent termination of translation at the final Pro codon of 2A. Subsequently, translation 'reinitiates' on the same codon, two individual proteins being generated from one open reading frame. Many 2A peptides have been identified, and they have a conserved C-terminal motif. Little similarity is present in the N-terminal portions of these peptides, which might suggest that these amino acids are not important in the 2A reaction. However, mutagenesis indicates that identity of the amino acid at nearly all positions of a single 2A peptide is important for activity. Each 2A may then represent a specific solution for positioning the conserved C-terminus within the peptidyl-transferase centre to promote recoding. Nascent 2A peptide:ribosome interactions are suggested to alter ribosomal fine structure to discriminate against prolyl-tRNA(Pro) and promote termination in the absence of a stop codon. Such structural modifications may account for our observation that replacement of the final Pro codon of 2A with any stop codon both stalls ribosome processivity and inhibits nascent chain release.     

The codon specificity of eubacterial release factors is determined by the sequence and size of the recognition loop

The two codon-specific eubacterial release factors (RF1: UAA/UAG and RF2: UAA/UGA) have specific tripeptide motifs (PXT/SPF) within an exposed recognition loop shown in recent structures to interact with stop codons during protein synthesis termination. The motifs have been inferred to be critical for codon specificity, but this study shows that they are insufficient to determine specificity alone. Swapping the motifs or the entire loop between factors resulted in a loss of codon recognition rather than a switch of codon specificity. From a study of chimeric eubacterial RF1/RF2 recognition loops and an atypical shorter variant in Caenorhabditis elegans mitochondrial RF1 that lacks the classical tripeptide motif PXT, key determinants throughout the whole loop have been defined. It reveals that more than one configuration of the recognition loop based on specific sequence and size can achieve the same desired codon specificity. This study has provided unexpected insight into why a combination of the two factors is necessary in eubacteria to exclude recognition of UGG as stop.     

Class-1 polypeptide chain release factors are structurally and functionally similar to suppressor tRNAs and comprise different structural-functional families of prokaryotic/mitochondrial and eukaryotic/archaebacterial factors

Class-1 polypeptide chain release factors (RF) induce peptidyl-tRNA hydrolysis in the ribosome if any of the three stop codons encounters the ribosomal A site. We have shown earlier that all factors of this class possess a common functionally essential motif GGQ. In this study we analyzed the primary structures of all known class-1 factors taken from the data banks together with the experimental data available on their structural and functional organization. The following conclusions were drawn. 1. Amino acid sequences of eukaryotic and archaebacterial factors (eRF1 and aRF1, respectively) show high similarity. This suggests the potential ability of eRF1 to function in archaebacterial and aRF1 in eukaryotic ribosomes, and points to their origin from a common ancestor. 2. Primary structures of class-1 release factors from prokaryotes and enkaryotic mitochondria show no statistically significant similarity with archaebacterial and cytoplasmic eukaryotic release factors, except for a common motif GGQ. This confirms our earlier conclusion (Nature, 1994, vol. 372, pp. 701–703) and contradicts the hypothesis of Itoet al. (Proc. Natl. Acad. Sci. USA, 1996, vol. 93, pp. 5443–5448) about structural similarity of all class-1 release factors. 3. All the eRF1/aRF1 recognizing three stop codons have a common motif NIKs that is absent from eubacterial RF1 and RF2, each of which is able to recognize two stop codons of the three. We suppose that the function of the NIKs motif is to fix the proper orientation of eRF1/aRF1 at the ribosome. 4. The domain structure and functional properties of eRF1/aRF1 point to the similarity of these factors with suppressor tRNAs as suggested long ago, and also semblance with aminoacyl-tRNA synthetases. 5. Considering that peptidyl-tRNA is fixed at the ribosomal P site while the stop codon and termination factor are at the A site, it may be presumed that the distance between the functionally essential motifs NIKs and GGQS in eRF1/aRF1 should approximately correspond to the distance between the anticodon and the aminoacyl end of aminoacyl-tRNA located at the ribosomal A site.     

Structural basis for translation termination by archaeal RF1 and GTP-bound EF1�� 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.     

Release factor binding to ribosome requires an intact 16 S rRNA 3' terminus.

Cloacin DF12 cleavage of Escherichia coli f[3H]MettRNA-AUG-ribosome complexes affects this substrate for in vitro peptide chain termination. Codon-directed release factors' (RF) 1 and 2 release of f[3H]methionine is inhibited by cloacin. Since cloacin inhibits RF1 and -2 binding to ribosomes but not RF-directed f[3H]methionine release from f[3H]met-tRNA-AUG-ribosome complexes when reactions contain 20% ethanol, we conclude that cloacin DF 13 inhibits formation of the termination codon recognition complex. Thus, cleavage of the 3'-OH 49-nucleotide sequence of the 16 S rRNA perturbs the codon-directed binding of RF to ribosomes.