The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure

Bacterial class I release factors (RFs) are seen by cryo-electron microscopy (cryo-EM) to span the distance between the ribosomal decoding and peptidyl transferase centers during translation termination. The compact conformation of bacterial RF1 and RF2 observed in crystal structures will not span this distance, and large structural rearrangements of RFs have been suggested to play an important role in termination. We have collected small-angle X-ray scattering (SAXS) data from E. coli RF1 and from a functionally active truncated RF1 derivative. Theoretical scattering curves, calculated from crystal and cryo-EM structures, were compared with the experimental data, and extensive analyses of alternative conformations were made. Low-resolution models were constructed ab initio, and by rigid-body refinement using RF1 domains. The SAXS data were compatible with the open cryo-EM conformation of ribosome bound RFs and incompatible with the crystal conformation. These conclusions obviate the need for assuming large conformational changes in RFs during termination.     

Diverse bacterial genomes encode an operon of two genes, one of which is an unusual class-I release factor that potentially recognizes atypical mRNA signals other than normal stop codons

Background  

While all codons that specify amino acids are universally recognized by tRNA molecules, codons signaling termination of translation are recognized by proteins known as class-I release factors (RF). In most eukaryotes and archaea a single RF accomplishes termination at all three stop codons. In most bacteria, there are two RFs with overlapping specificity, RF1 recognizes UA(A/G) and RF2 recognizes U(A/G)A.     

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.     

八肋游仆虫两类释放因子的相互作用

从八肋游仆虫中克隆到两类释放因子基因Eo-eRFI和Eo-eRF3。在Eo-eRF3基因的阅读框中有3个通用的终止密码子UGA,在此编码半胱氨酸。为了研究两类释放因子的相互作用,用PCR的方法对3个位点进行了定点突变,将UGA突变为通用的编码半胱氨酸的密码子UGU。突变结果经测序确认后,在大肠杆菌中获得全长Eo-eRF3的正确表达。在此基础上,构建酵母双杂交重组质粒,用该系统检测了游仆虫两类释放因子的相互作用。结果显示,两类释放因子在生物体内形成复合体,从而在较原始的真核生物中,证实了两类释放因子的相互作用关系。     

Distinct Roles for Release Factor 1 and Release Factor 2 in Translational Quality Control

In bacteria, stop codons are recognized by two similar class 1 release factors, release factor 1 (RF1) and release factor 2 (RF2). Normally, during termination, the class 2 release factor 3 (RF3), a GTPase, functions downstream of peptide release where it accelerates the dissociation of RF1/RF2 prior to ribosome recycling. In addition to their canonical function in termination, both classes of release factor are also involved in a post peptidyl transfer quality control (post PT QC) mechanism where the termination factors recognize mismatched (i.e. error-containing) ribosome complexes and promote premature termination. Here, using a well defined in vitro system, we explored the role of release factors in canonical termination and post PT QC. As reported previously, during canonical termination, RF1 and RF2 recognize stop codons in a similar manner, and RF3 accelerates their rate of dissociation. During post PT QC, only RF2 (and not RF1) effectively binds to mismatched ribosome complexes; and whereas the addition of RF3 to RF2 increased its rate of release on mismatched complexes, the addition of RF3 to RF1 inhibited its rate of release but increased the rate of peptidyl-tRNA dissociation. Our data strongly suggest that RF2, in addition to its primary role in peptide release, functions as the principle factor for post PT QC.     

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.     

Structure of a human translation termination complex

In contrast to bacteria that have two release factors, RF1 and RF2, eukaryotes only possess one unrelated release factor eRF1, which recognizes all three stop codons of the mRNA and hydrolyses the peptidyl-tRNA bond. While the molecular basis for bacterial termination has been elucidated, high-resolution structures of eukaryotic termination complexes have been lacking. Here we present a 3.8 Å structure of a human translation termination complex with eRF1 decoding a UAA(A) stop codon. The complex was formed using the human cytomegalovirus (hCMV) stalling peptide, which perturbs the peptidyltransferase center (PTC) to silence the hydrolysis activity of eRF1. Moreover, unlike sense codons or bacterial stop codons, the UAA stop codon adopts a U-turn-like conformation within a pocket formed by eRF1 and the ribosome. Inducing the U-turn conformation for stop codon recognition rationalizes how decoding by eRF1 includes monitoring geometry in order to discriminate against sense codons.     

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.     

Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon

During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate stop codons, at resolutions of 5.9 Angstrom and 6.7 Angstrom, respectively. The structures reveal details of interactions of the factors with the ribosome and mRNA, including elements previously implicated in decoding and peptide release. They also shed light on conformational changes both in the factors and in the ribosome during termination. Differences seen in the interaction of RF1 and RF2 with the L11 region of the ribosome allow us to rationalize previous biochemical data. Finally, this work demonstrates the feasibility of crystallizing ribosomes with bound factors at a defined state along the translational pathway.     

Nonstandard genetic codes and translation termination

Genetic code is not universal. Various nonstandard versions of the code are known for some mitochondrial, prokaryotic, and eukaryotic genomes. The most common deviation is stop codon reassignment; i.e., a stop codon is decoded as a sense codon rather than as a signal for translation termination. Class 1 release factors (RFs: prokaryotic RF1 and RF2 and eukaryotic eRF1) recognize the stop codons and induce hydrolysis of peptidyl-tRNA in the ribosome. The specificity of class 1 RFs changes in organisms with a nonstandard code. The rare amino acids selenocysteine and pyrrolysine utilize essentially different decoding strategies. The review considers several hypotheses of the origin of nonstandard genetic codes. A new hypothesis is advanced, assuming a change in the specificity of class 1 RFs as a starting point for stop codon reassignment.     

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.     

Quantitative analysis of in vivo ribosomal events at UGA and UAG stop codons.

An in vivo translation assay system has been designed to measure, in one and the same assay, the three alternatives for a ribosome poised at a stop codon (termination, read-through and frameshift). A quantitative analysis of the competition has been done in the presence and absence of release factor (RF) mutants, nonsense suppressors and an upstream Shine-Dalgarno-like sequence. The ribosomal +1 frameshift product is measurable when the stop codon is decoded by wild-type or mutant RF (prf A1 or prf B2) and also in the presence of competing suppressor tRNAs. Frameshift frequency appears to be influenced by RF activity. The amount of frameshift product decreases in the presence of competing suppressor tRNAs, however, this decrease is not in proportion to the corresponding increase in the suppression product. Instead, there is an increase in the total amount of protein expressed from the gene, perhaps due to the purging of queued ribosomes. Mutated RFs reduce the total output of the reporter gene by reducing the amount of all three protein products. The nascent peptide has earlier been shown to influence the translation termination process by interacting with the RFs. At 42 degrees C in a temperature-sensitive RF mutant strain, protein measurements indicate that the nascent peptide seems to influence the binding efficiencies of the RFs.     

Release factors 2 from Escherichia coli and Thermus thermophilus: structural, spectroscopic and microcalorimetric studies

Prokaryotic class I release factors (RFs) respond to mRNA stop codons and terminate protein synthesis. They interact with the ribosomal decoding site and the peptidyl-transferase centre bridging these 75 A distant ribosomal centres. For this an elongated RF conformation, with partially unfolded core domains II.III.IV is required, which contrasts the known compact RF crystal structures. The crystal structure of Thermus thermophilus RF2 was determined and compared with solution structure of T. thermophilus and Escherichia coli RF2 by microcalorimetry, circular dichroism spectroscopy and small angle X-ray scattering. The structure of T. thermophilus RF2 in solution at 20 degrees C is predominantly compact like the crystal structure. Thermodynamic analysis point to an initial melting of domain I, which is independent from the melting of the core. The core domains II.III.IV melt cooperatively at the respective physiological temperatures for T. thermophilus and E. coli. Thermodynamic analyses and the X-ray scattering results for T. thermophilus RF2 in solution suggest that the compact conformation of RF2 resembles a physiological state in absence of the ribosome.     

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.     

Recognition of the amber UAG stop codon by release factor RF1

We report the crystal structure of a termination complex containing release factor RF1 bound to the 70S ribosome in response to an amber (UAG) codon at 3.6‐Å resolution. The amber codon is recognized in the 30S subunit‐decoding centre directly by conserved elements of domain 2 of RF1, including T186 of the PVT motif. Together with earlier structures, the mechanisms of recognition of all three stop codons by release factors RF1 and RF2 can now be described. Our structure confirms that the backbone amide of Q230 of the universally conserved GGQ motif is positioned to contribute directly to the catalysis of the peptidyl‐tRNA hydrolysis reaction through stabilization of the leaving group and/or transition state. We also observe synthetic‐negative interactions between mutations in the switch loop of RF1 and in helix 69 of 23S rRNA, revealing that these structural features interact functionally in the termination process. These findings are consistent with our proposal that structural rearrangements of RF1 and RF2 are critical to accurate translation termination.     

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.     

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.     

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.     

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.     

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.