Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.     

How does <Emphasis Type="Italic">Euplotes</Emphasis> translation termination factor eRF1 fail to recognize the UGA stop codon?

Class 1 eukaryotic release factor 1 (eRF1) recognizes all three stop codons (UAA, UAG, and UGA) in standard-code organisms. In some ciliates with variant genetic codes, one or two stop codons are used to encode amino acids and are not recognized by eRF1; e.g., UAA and UAG are reassigned to Gln in Stylonychia and UGA is reassigned to Cys in Euplotes. Stop codon recognition is due to the N-terminal domain of eRF1 in standard-code organisms. Since variant-code ciliates most likely originate from universal-code ancestors, the N-domain sequence of their eRF1 was assumed to harbor the residues that are responsible for the changes in stop codon recognition specificity. To identify the N-domain regions determining the UGA-only specificity of Euplotes aediculatus eRF1, chimeric proteins were constructed by swapping various N-domain fragments of the E. aediculatus for their human counterparts; the MC domain was from human eRF1. Functional analysis of the chimeric eRF1 in vivo revealed two regions (residues 38–50 and 123–145) restricting the E. aediculatus eRF1 specificity to UAR. The change in stop codon recognition specificity of eRF1 was regarded as the first step in the origin of the variant genetic code in ciliates.     

Class I release factors in ciliates with variant genetic codes

In eukaryotes with the universal genetic code a single class I release factor (eRF1) most probably recognizes all stop codons (UAA, UAG and UGA) and is essential for termination of nascent peptide synthesis. It is well established that stop codons have been reassigned to amino acid codons at least three times among ciliates. The codon specificities of ciliate eRF1s must have been modified to accommodate the variant codes. In this study we have amplified, cloned and sequenced eRF1 genes of two hypotrichous ciliates, Oxytricha trifallax (UAA and UAG for Gln) and Euplotes aediculatus (UGA for Cys). We also sequenced/identified three protist and two archaeal class I RF genes to enlarge the database of eRF1/aRF1s with the universal code. Extensive comparisons between universal code eRF1s and those of Oxytricha, Euplotes, and Tetrahymena which represent three lineages that acquired variant codes independently, provide important clues to identify stop codon-binding regions in eRF1. Domain 1 in the five ciliate eRF1s, particularly the TASNIKS heptapeptide and its adjacent region, differs significantly from domain 1 in universal code eRF1s. This observation suggests that domain 1 contains the codon recognition site, but that the mechanism of eRF1 codon recognition may be more complex than proposed by Nakamura et al. or Knight and Landweber.     

Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1

In eukaryotic ribosomes, termination of translation is triggered by class 1 polypeptide release factor, eRF1. In organisms with a universal code, eRF1 responds to three stop codons, whereas, in ciliates with variant codes, only one or two codon(s) remain(s) as stop signals. By mutagenesis of the Y-C-F minidomain of the N domain, we converted an omnipotent human eRF1 recognizing all three stop codons into a unipotent 'ciliate-like' UGA-only eRF1. The conserved Cys127 located in the Y-C-F minidomain plays a critical role in stop codon recognition. The UGA-only response has also been achieved by concomitant substitutions of four other amino acids located at the Y-C-F and NIKS minidomains of eRF1. We suggest that for eRF1 the stop codon decoding is of a non-linear (non-protein-anticodon) type and explores a combination of positive and negative determinants. We assume that stop codon recognition is profoundly different by eukaryotic and prokaryotic class 1 RFs.     

Isolation and expression of two genes encoding eukaryotic release factor 1 from Paramecium tetraurelia

Paramecium tetraurelia, like some other ciliate species, uses an alternative nuclear genetic code where UAA and UAG are translated as glutamine and UGA is the only stop codon. It has been postulated that the use of stop codons as sense codons is dependent on the presence of specific tRNAs and on modification of eukaryotic release factor one (eRF1), a factor involved in stop codon recognition during translation termination. We describe here the isolation and characterisation of two genes, eRF1-a and eRF1 b, coding for eRF1 in P. tetraurelia. The two genes are very similar, both in genomic organization and in sequence, and might result from a recent duplication event. The two coding sequences are 1,314 nucleotides long, and encode two putative proteins of 437 amino acids with 98.5% identity. Interestingly, when compared with the eRF1 sequences either of ciliates having the same variant genetic code, or of other eukaryotes, the eRF1 of P. tetraurelia exhibits significant differences in the N-terminal region, which is thought to interact with stop codons. We discuss here the consequences of these changes in the light of recent models proposed to explain the mechanism of stop codon recognition in eukaryotes. Besides, analysis of the expression of the two genes by Northern blotting and primer extension reveals that these genes exhibit a differential expression during vegetative growth and autogamy.     

四膜虫eRF1识别终止密码子的功能结构域

原生动物的一些纤毛虫中终止密码子发生重分配现象,将1个或2个终止密码子翻译为氨基酸.目前对这一现象的发生机制仍无合理解释．近年来,对蛋白质合成终止过程中肽链释放因子(eukaryotic polypeptide release factor, eRF)结构和功能的深入研究,为揭示终止密码子的重分配机制提供了重要的线索．本实验以具有终止密码子识别特异性的四膜虫Tt-eRF1为研究对象,将其中与密码子识别有关的GTx、NIKS和Y-C-F关键模体(motif) 引入识别通用终止密码子的酵母Sc=eRF1中,构建成各种嵌合体eRF1．利用双荧光素酶报告系统和细胞活性实验,分析关键模体及其周边的氨基酸对eRF1识别终止密码子性质的影响．结果表明,GTx和NIKS模体一定程度上决定eRF1识别终止密码子第1位碱基U和第2位碱基A;Y-C-F模体决定eRF1识别终止密码子UGA的第2位碱基G．模体内及其相邻氨基酸定点突变分析进一步支持以上结果．本研究推测,eRF1在进化过程中一些关键模体结构的改变决定其识别终止密码子的特异性,只能识别3个终止密码子中的1个或2个．随后,由于tRNA基因的突变产生阻抑性tRNA,促成终止密码子在原生动物纤毛虫中的重新分配．     

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.     

Molecular mechanism of stop codon recognition by eRF1: a wobble hypothesis for peptide anticodons

We propose that the amino acid residues 57/58 and 60/61 of eukaryotic release factors (eRF1s) (counted from the N-terminal Met of human eRF1) are responsible for stop codon recognition in protein synthesis. The proposal is based on amino acid exchanges in these positions in the eRF1s of two ciliates that reassigned one or two stop codons to sense codons in evolution and on the crystal structure of human eRF1. The proposed mechanism of stop codon recognition assumes that the amino acid residues 57/58 interact with the second and the residues 60/61 with the third position of a stop codon. The fact that conventional eRF1s recognize all three stop codons but not the codon for tryptophan is attributed to the flexibility of the helix containing these residues. We suggest that the helix is able to assume a partly relaxed or tight conformation depending on the stop codon recognized. The restricted codon recognition observed in organisms with unconventional eRF1s is attributed mainly to the loss of flexibility of the helix due to exchanged amino acids.     

The ciliate Euplotes octocarinatus expresses two polypeptide release factors of the type eRF1

Amplification of macronuclear DNA of the ciliate Euplotes octocarinatus revealed the presence of two genes encoding putative polypeptide release factors (RFs) of the codon specific class-I type. They are named eRF1a and eRF1b, respectively. cDNA amplification revealed that both eRF1 genes are expressed. Determination of their copy numbers showed that they are similarly amplified to a level of about 27,000. The deduced protein sequences of the two genes are 57 and 58% identical with human eRF1 and 79% identical to each other. The gene encoding eRF1b possesses three in-frame UGA codons. This codon is known to encode cysteine in Euplotes; only UAA and UAG are used as stop codons in this organism. The primary structure of the two release factors is analyzed and compared with the primary structure of other eukaryotic release factors including the one of Tetrahymena thermophila which uses only UGA as a stop codon. eRF1a and eRF1b of Euplotes as well as eRF1 of Tetrahymena differ from human eRF1 and other class-I release factors of eukaryotes in a domain recently proposed to be responsible for codon recognition. Based on the changes which we observe in this region and the differential use of the stop codons in these two ciliates we predict the amino acids participating in stop codon recognition in eRF1 release factors.     

Non-standard genetic codes and translation termination

Genetic code is not universal. Various non-standard versions of the code were found in mitochondrial, prokaryotic and eukaryotic genomes. Stop codons are used to signal the ribosome stop translation of the coding sequence and prone to reassignment to sense codons. Class-1 termination factors recognize stop codons and promote hydrolysis of the peptidyl-tRNA in ribosome (RF1, RF2 in prokaryotes and eRF1 in eukaryotes). The class-1 factor termination specificity is changed in non-standart codes organisms. Pyrrolysine and selenocysteine use dissimilar decoding strategies. The various non-standart code origin hypotheses are described. It was proposed that specificity alteration of the class-1 release factor was a starting point for stop codon reassignment.     

第一类肽链释放因子C端结构域参与调控终止密码子的识别过程

真核生物蛋白质翻译终止过程中,第一类肽链释放因子（eukaryotic polypeptide release factor, eRF1）利用其N端结构域识别终止密码子。eRF1的N结构域中的GTS、NIKS和YxCxxxF模体对于终止密码子的识别发挥重要作用。但至目前为止,eRF1识别终止密码子的机制,尤其是对于终止密码子的选择性识别机制仍不清楚。我们构建了四膜虫（Tetrahymena thermophilia）eRF1的N端结构域与酿酒酵母（Saccharomyces cerevisiae）或裂殖酵母（Schizosaccharomyces pombe）eRF1的M和C结构域组成的杂合eRF1,即Tt/Sc eRF1 和Tt/Sp eRF1。双荧光素酶检测结果证实,两种杂合eRF1在细胞中识别终止密码子的活性具有显著差异。Tt/Sc eRF1仅识别UGA密码子,与四膜虫eRF1一致,具有密码子识别特异性;而Tt/Sp eRF1可以识别3个终止密码子,无密码子识别特异性。为解释这一现象,将Sp eRF1的C结构域中的1个关键的小结构域中的氨基酸进行突变,与Sc eRF1相应位点的氨基酸一致。分析结果显示,突变体Tt/Sp eRF1识别密码子UAA和UAG的性质发生显著变化,说明第一类肽链释放因子的C端结构域参与了终止密码子的识别过程。这提示,四膜虫eRF1识别终止密码子的特异性可能依赖于eRF1分子内的结构域间相互作用。本研究结果为揭示肽链释放因子识别终止密码子的分子机制提供了数据支持。     

Stop codon selection in eukaryotic translation termination: comparison of the discriminating potential between human and ciliate eRF1s

During eukaryotic translation termination, eRF1 responds to three stop codons. However, in ciliates with variant genetic codes, only one or two codons function as a stop signal. To localize the region of ciliate eRF1 implicated in stop codon discrimination, we have constructed ciliate-human hybrid eRF1s by swapping regions of human eRF1 for the equivalent region of ciliate Euplotes eRF1. We have examined the formation of a cross-link between recombinant eRF1s and mRNA analogs containing the photoactivable 4-thiouridine (s(4)U) at the first position of stop and control sense codons. With human eRF1, this cross-link can be detected only when either stop or UGG codons are located in the ribosomal A site. Here we show that the cross-link of the Euplotes-human hybrid eRF1 is restricted to mRNAs containing UAG and UAA codons, and that the entire N-terminal domain of Euplotes eRF1 is involved in discriminating against UGA and UGG. On the basis of these results, we discuss the steps of the selection process that determine the accuracy of stop codon recognition in eukaryotes.     

Connection between stop codon reassignment and frequent use of shifty stop frameshifting

Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recently demonstrated that Euplotes octocarinatus peptide release factor eRF1 ignores UGA termination codons while continuing to recognize UAA and UAG. Here we show that both the Tetrahymena thermophila and E. octocarinatus eRF1 factors allow efficient frameshifting at all three termination codons, suggesting that UGA redefinition also impaired UAA/UAG recognition. Mutations of the Euplotes factor restoring a phylogenetically conserved motif in eRF1 (TASNIKS) reduced programmed frameshifting at all three termination codons. Mutation of another conserved residue, Cys124, strongly reduces frameshifting at UGA while actually increasing frameshifting at UAA/UAG. We will discuss these results in light of recent biochemical characterization of these mutations.     

How translation termination factor eRF1 Euplotes does not recognise UGA stop codon

In universal-code eukaryotes, a single class-1 translation termination factor eRF1 decodes all three stop codons, UAA, UAG, and UGA. In some ciliates with variant genetic codes one or two stop codons are used to encode amino acid(s) and are not recognized by eRF1. In Stylonychia, UAG and UAA codons are reassigned as glutamine codons, and in Euplotes, UGA is reassigned as cysteine codon. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of eRF1. Because variant-code ciliates most likely evolved from universal code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. To find out amino acid residues which confer UAR-only specificity to Euplotes aediculatus eRF1, eRFI chimeras were constructed by swapping eRF1 E. aediculatus N-terminal domain sequences with the matching ones from the human protein. In these chimeras the MC-domain was from human eRF1. Functional analysis of these chimeric eRFI highlighted the crucial role of the two regions (positions 38-50 and 123-145) in the N-terminal domain of E. aediculatus eRF1 that restrict E. aediculatus eRF1 specificity toward UAR codons. Possibly, restriction of eRF1 specificity to UAR codons might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UGA to sense codons.     

八肋游仆虫第二类肽链释放因子核定位信号分析

蛋白质合成终止过程中肽链释放因子负责终止密码子的识别.真核生物第二类肽链释放因子（eRF3）是一类GTP酶,协助第一类肽链释放因子（eRF1）识别终止密码子和水解肽酰 tRNA酯键.之前的研究表明,两类肽链释放因子在细胞核中发挥功能,参与蛋白质合成和纺锤体的组装.本研究根据软件预测结果,构建了一系列八肋游仆虫eRF3的截短型突变体,分析在其N端是否存在引导eRF3的核定位信号.结果表明,在eRF3的N端有两个区域（NLS1:23-36 aa 和 NLS2: 236-272 aa）可以引导eRF3进入细胞核中,而且这两个区域具有典型的核定位信号的氨基酸序列特征. eRF3的核定位与其作为一种穿梭蛋白的功能相一致,即参与细胞有丝分裂纺锤体的形成和无义介导的mRNA降解途径.     

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.     

Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes

The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.     

A single amino acid change of translation termination factor eRF1 switches between bipotent and omnipotent stop-codon specificity

In eukaryotes a single class-1 translation termination factor eRF1 decodes the three stop codons: UAA, UAG and UGA. Some ciliates, like Euplotes, have a variant code, and here eRF1s exhibit UAR-only specificity, whereas UGA is reassigned as a sense codon. Since eukaryote eRF1 stop-codon recognition is associated with its N-terminal domain, structural features should exist in the N domain of ciliate eRF1s that restrict their stop-codon specificity. Using an in vitro reconstituted eukaryotic translation system we demonstrate here that a chimeric eRF1 composed of the N domain of Euplotes aediculatus eRF1 fused to the MC domains of human eRF1 exhibits UAR-only specificity. Functional analysis of eRF1 chimeras constructed by swapping Euplotes N domain sequences with the cognate regions from human eRF1 as well as site-directed mutagenesis of human eRF1 highlighted the crucial role of the alanine residue in position 70 of E. aediculatus eRF1 in restricting UGA decoding. Switching the UAR-only specificity of E. aediculatus eRF1 to omnipotent mode is due to a single point mutation. Furthermore, we examined the influence of eRF3 on the ability of chimeric and mutant eRF1s to induce peptide release in response to different stop codons.     

Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition

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.