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.     

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.     

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.     

Molecular basis for bacterial class I release factor methylation by PrmC

Class I release factors bind to ribosomes in response to stop codons and trigger peptidyl-tRNA hydrolysis at the P site. Prokaryotic and eukaryotic RFs share one motif: a GGQ tripeptide positioned in a loop at the end of a stem region that interacts with the ribosomal peptidyl transferase center. The glutamine side chain of this motif is specifically methylated in both prokaryotes and eukaryotes. Methylation in E. coli is due to PrmC and results in strong stimulation of peptide chain release. We have solved the crystal structure of the complex between E. coli RF1 and PrmC bound to the methyl donor product AdoHCy. Both the GGQ domain (domain 3) and the central region (domains 2 and 4) of RF1 interact with PrmC. Structural and mutagenic data indicate a compact conformation of RF1 that is unlike its conformation when it is bound to the ribosome but is similar to the crystal structure of the protein alone.     

Common and specific amino acid residues in the prokaryotic polypeptide release factors RF1 and RF2: possible functional implications

Termination of protein synthesis is promoted in ribosomes by proper stop codon discrimination by class 1 polypeptide release factors (RFs). A large set of prokaryotic RFs differing in stop codon specificity, RF1 for UAG and UAA, and RF2 for UGA and UAA, was analyzed by means of a recently developed computational method allowing identification of the specificity-determining positions (SDPs) in families composed of proteins with similar but not identical function. Fifteen SDPs were identified within the RF1/2 superdomain II/IV known to be implicated in stop codon decoding. Three of these SDPs had particularly high scores. Five residues invariant for RF1 and RF2 [invariant amino acid residues (IRs)] were spatially clustered with the highest-scoring SDPs that in turn were located in two zones within the SDP/IR area. Zone 1 (domain II) included PxT and SPF motifs identified earlier by others as 'discriminator tripeptides'. We suggest that IRs in this zone take part in the recognition of U, the first base of all stop codons. Zone 2 (domain IV) possessed two SDPs with the highest scores not identified earlier. Presumably, they also take part in stop codon binding and discrimination. Elucidation of potential functional role(s) of the newly identified SDP/IR zones requires further experiments.     

The function, structure and regulation of E. coli peptide chain release factors

The termination of protein synthesis in Escherichia coli depends upon the soluble protein factors RF1 or RF2. RF1 catalyzes UAG and UAA dependent termination, while RF2 catalyzes UGA and UAA dependent termination. The proteins have been purified to homogeneity, their respective genes isolated, and their primary structures deduced from the DNA sequences. The sequences reveal considerable conserved homology, presumably reflecting functional similarities and a common ancestral origin. The RFs are encoded as single copy genes on the bacterial chromosome. RF2 exhibits autogenous regulation in an in vitro translation system. The mechanism of autoregulation appears to be an in-frame UGA stop codon that requires a 1+ frameshift for the continued synthesis of the protein. Frameshifting prior to the inframe stop codon occurs at a remarkably high frequency by an unknown mechanism. Future studies will be directed at understanding how RFs interact with the ribosomal components, and further defining the mechanism of RF2 frameshifting.     

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.     

Release Factor One Is Nonessential in Escherichia coli

Recoding a stop codon to an amino acid may afford orthogonal genetic systems for biosynthesizing new protein and organism properties. Although reassignment of stop codons has been found in extant organisms, a model organism is lacking to investigate the reassignment process and to direct code evolution. Complete reassignment of a stop codon is precluded by release factors (RFs), which recognize stop codons to terminate translation. Here we discovered that RF1 could be unconditionally knocked out from various Escherichia coli stains, demonstrating that the reportedly essential RF1 is generally dispensable for the E. coli species. The apparent essentiality of RF1 was found to be caused by the inefficiency of a mutant RF2 in terminating all UAA stop codons; a wild type RF2 was sufficient for RF1 knockout. The RF1-knockout strains were autonomous and unambiguously reassigned UAG to encode natural or unnatural amino acids (Uaas) at multiple sites, affording a previously unavailable model for studying code evolution and a unique host for exploiting Uaas to evolve new biological functions.     

Indirect regulation of translational termination efficiency at highly expressed genes and recoding sites by the factor recycling function of Escherichia coli release factor RF3.

Prokaryotic release factor RF3 is a stimulatory protein that increases the rate of translational termination by the decoding release factors RF1 and RF2. The favoured model for RF3 function is the recycling of RF1 and RF2 after polypeptide release by displacing the factors from the ribosome. In this study, we have demonstrated that RF3 also plays an indirect role in the decoding of stop signals of highly expressed genes and recoding sites by accentuating the influence of the base following the stop codon (+4 base) on termination signal strength. The efficiency of decoding strong stop signals (e.g. UAAU and UAAG) in vivo is markedly improved with increased RF3 activity, while weak signals (UGAC and UAGC) are only modestly affected. However, RF3 is not responsible for the +4 base influence on termination signal strength, since prfC- strains lacking the protein still exhibit the same qualitative effect. The differential effect of RF3 at stop signals can be mimicked by modest overexpression of decoding RF. These findings can be interpreted according to current views of RF3 as a recycling factor, which functions to maintain the concentration of free decoding RF at stop signals, some of which are highly responsive to changes in RF levels.     

Release factors eRF1 and RF2: a universal mechanism controls the large conformational changes

Class I release factors 1 and 2 (RF1 and RF2) terminate protein synthesis by recognizing stop codons on the mRNA via their conserved amino acid motifs (NIKS in eRF1 and SPF in RF2) and by the conserved tripeptide (GGQ) interactions with the ribosomal peptidyltransferase center. Crystal structures of eRF1 and RF2 do not fit their ribosomal binding pocket (approximately 73 angstroms). Cryoelectron microscopy indicates large conformational changes in the ribosome-bound RF2. Here, we investigate the conformational dynamics of the eRF1 and RF2 using molecular dynamics simulation, structural alignment, and electrostatic analysis of domain interactions. We show that relaxed eRF1 has a shape remarkably similar to the ribosome-bound RF2 observed by cryoelectron microscopy. The similarity between the two release factors is as good as between elongation factor G and elongation factor Tu-guanosine-5'(beta,gamma-imido)triphosphate-tRNA. Further, the conformational transitions and dynamics of eRF1 and RF2 between the free and ribosome-bound states are most likely controlled by protonation of conserved histidines. For eRF1, the distance between the NIKS and GGQ motifs shrinks from 97.5 angstroms in the crystal to 70-80 angstroms. For RF2, the separation between SPF and GGQ elongates from 32 angstroms in the crystal to 50 angstroms. Coulombic interaction strongly favors the open conformation of eRF1; however, solvation and histidine protonation modulate the domain interactions, making the closed conformation of eRF1 more accessible. Thus, RF1 and RF2 function like molecular machines, most likely fueled by histidine protonation. The unified conformational control and the shapes of eRF1 and RF2 support the proposition that the termination of protein synthesis involves similar mechanisms across species.     

Variation in Release Factor Abundance Is Not Needed to Explain Trends in Bacterial Stop Codon Usage

In bacteria stop codons are recognized by one of two class I release factors (RF1) recognizing TAG, RF2 recognizing TGA, and TAA being recognized by both. Variation across bacteria in the relative abundance of RF1 and RF2 is thus hypothesized to select for different TGA/TAG usage. This has been supported by correlations between TAG:TGA ratios and RF1:RF2 ratios across multiple bacterial species, potentially also explaining why TAG usage is approximately constant despite extensive variation in GC content. It is, however, possible that stop codon trends are determined by other forces and that RF ratios adapt to stop codon usage, rather than vice versa. Here, we determine which direction of the causal arrow is the more parsimonious. Our results support the notion that RF1/RF2 ratios become adapted to stop codon usage as the same trends, notably the anomalous TAG behavior, are seen in contexts where RF1:RF2 ratios cannot be, or are unlikely to be, causative, that is, at 3′untranslated sites never used for translation termination, in intragenomic analyses, and across archaeal species (that possess only one RF1). We conclude that specifics of RF biology are unlikely to fully explain TGA/TAG relative usage. We discuss why the causal relationships for the evolution of synonymous stop codon usage might be different from those affecting synonymous sense codon usage, noting that transitions between TGA and TAG require two-point mutations one of which is likely to be deleterious.     

Stop codon recognition and interactions with peptide release factor RF3 of truncated and chimeric RF1 and RF2 from Escherichia coli

Release factors RF1 and RF2 recognize stop codons present at the A-site of the ribosome and activate hydrolysis of peptidyl-tRNA to release the peptide chain. Interactions with RF3, a ribosome-dependent GTPase, then initiate a series of reactions that accelerate the dissociation of RF1 or RF2 and their recycling between ribosomes. Two regions of Escherichia coli RF1 and RF2 were identified previously as involved in stop codon recognition and peptidyl-tRNA hydrolysis. We show here that removing the N-terminal domain of RF1 or RF2 or exchanging this domain between the two factors does not affect RF specificity but has different effects on the activity of RF1 and RF2: truncated RF1 remains highly active and able to support rapid cell growth, whereas cells with truncated RF2 grow only poorly. Transplanting a loop of 13 amino acid residues from RF2 to RF1 switches the stop codon specificity. The interaction of the truncated factors with RF3 on the ribosome is defective: they fail to stimulate guanine nucleotide exchange on RF3, recycling is not stimulated by RF3, and nucleotide-free RF3 fails to stabilize the binding of RF1 or RF2 to the ribosome. However, the N-terminal domain seems not to be required for the expulsion of RF1 or RF2 by RF3:GTP.     

Molecular recognition and catalysis in translation termination complexes

When the ribosome machinery reaches a stop codon in the mRNA, protein synthesis stops, and nascent polypeptide release is catalysed by class-I release factors (RFs); class-II RFs then promote the release of class-I RFs. Cryo electron microscopy structures of termination complexes and crystal structures of isolated factors have provided insights into key concepts such as bridging of active sites on the ribosome, and conformational changes that regulate the termination process. Recent crystal structures of the four possible functional ribosome complexes that contain the class-I RFs and the three stop codons have uncovered the molecular mechanisms by which RF1/RF2 (i) both recognise UAA, but discriminate specifically between UAG and UGA, and (ii) catalyse peptide release. Moreover, ongoing research also promises to reveal the structure-function relations of class-II RFs.     

Immunochemical determination of cellular content of translation release factor RF4 in Escherichia coli

The biosynthesis of proteins in prokaryotes is terminated when a stop codon is present in the A-site of the 70S ribosomal complex. Four different translation termination factors are known to participate in the termination process. Release factor RF1 and RF2 are responsible for the recognition of the stop codons, and RF3 is known to accelerate the overall termination process. Release factor RF4 is a protein involved in the release of the mRNA and tRNA from the ribosomal complex. Furthermore, RF4 is involved in the proofreading in the elongation step of protein biosynthesis. The cellular contents of RF1, RF2, and RF3 were determined earlier. Here we report the cellular content of RF4 in Escherichia coli to be approximately 16,500 molecules per cell. The cells were grown in a rich medium and harvested in the beginning of the exponential growth phase. The quantifications were performed by using Western immunoblotting with radioactive iodinated streptavidin and biotinylated rabbit anti-mouse immunoglobulins plus a highly specific monoclonal antibody against RF4 as first antibody.     

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.     

Adenine and guanine recognition of stop codon is mediated by different N domain conformations of translation termination factor eRF1

Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125-131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3'-phosphate of UAA, while the same cross-linker at the 3'-phosphate of UAAA modified both the 26-28 and 67-73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines.     

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.     

Timing of GTP binding and hydrolysis by translation termination factor RF3

Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that—in contrast to a previous report—RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3–GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3–GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3–GTP–ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock.     

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.     

肽链释放因子识别终止密码子的机制

肽链释放因子(polypeptide release factor, RF)是参与细胞内蛋白质合成终止过程中新生肽链释放的一组重要的蛋白质,包括两类,即第一类肽链释放因子(classⅠrelease factor, RFⅠ)和第二类肽链释放因子(classⅡrelease factor, RFⅡ).关于第一类肽链释放因子识别终止密码子的机制和功能位点是目前分子细胞生物学领域的一个研究热点,第二类肽链释放因子作为一类GTP酶,在第一类肽链释放因子识别终止密码子和肽链释放过程中的协同作用也备受关注.近些年来,通过构建体内和体外的测活体系,对第一类肽链释放因子识别终止密码子的机制的研究取得了一些进展,提出了多种假说和模型,尤其是对第一类肽链释放因子的晶体结构及两类肽链释放因子复合体的空间结构的研究,为揭示真核生物细胞内蛋白质合成终止机制提供了直接的证据.