The invariant uridine of stop codons contacts the conserved NIKSR loop of human eRF1 in the ribosome

To unravel the region of human eukaryotic release factor 1 (eRF1) that is close to stop codons within the ribosome, we used mRNAs containing a single photoactivatable 4-thiouridine (s(4)U) residue in the first position of stop or control sense codons. Accurate phasing of these mRNAs onto the ribosome was achieved by the addition of tRNA(Asp). Under these conditions, eRF1 was shown to crosslink exclusively to mRNAs containing a stop or s(4)UGG codon. A procedure that yielded (32)P-labeled eRF1 deprived of the mRNA chain was developed; analysis of the labeled peptides generated after specific cleavage of both wild-type and mutant eRF1s maps the crosslink in the tripeptide KSR (positions 63-65 of human eRF1) and points to K63 located in the conserved NIKS loop as the main crosslinking site. These data directly show the interaction of the N-terminal (N) domain of eRF1 with stop codons within the 40S ribosomal subunit and provide strong support for the positioning of the eRF1 middle (M) domain on the 60S subunit. Thus, the N and M domains mimic the tRNA anticodon and acceptor arms, respectively.     

Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3'-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA(Phe), targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3'-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.     

Termination of protein synthesis

One of three mRNA codons — UAA, UAG and UGA — is used to signal to the elongating ribosome that translation should be terminated at this point. Upon the arrival of the stop codon at the ribosomal acceptor(A)-site, a protein release factor (RF) binds to the ribosome resulting in the peptidyl transferase centre of the ribosome switching to a hydrolytic function to remove the completed polypeptide chain from the peptidyl-tRNA bound at the adjacent ribosomal peptidyl(P)-site. In this review recent advances in our understanding of the mechanism of termination in the bacteriumEscherichia coli will be summarised, paying particular attention to the roles of 16S ribosomal RNA and the release factors RF-1, RF-2 and RF-3 in stop codon recognition. Our understanding of the translation termination process in eukaryotes is much more rudimentary with the identity of the single eukaryotic release factor (eRF) still remaining elusive. Finally, several examples of how the termination mechanism can be subverted either to expand the genetic code (e.g. selenocysteine insertion at UGA codons) or to regulate the expression of mammalian retroviral or plant viral genomes will be discussed.     

Eukaryote-specific motif of ribosomal protein S15 neighbors A site codon during elongation and termination of translation

The eukaryotic ribosomal protein S15 is a key component of the decoding site in contrast to its prokaryotic counterpart, S19p, which is located away from the mRNA binding track on the ribosome. Here, we determined the oligopeptide of S15 neighboring the A site mRNA codon on the human 80S ribosome with the use of mRNA analogues bearing perfluorophenyl azide-modified nucleotides in the sense or stop codon targeted to the 80S ribosomal A site. The protein was cross-linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of eRF1 in the experiments with mRNAs bearing stop codon. Digestion of modified S15 with various specific proteolytic agents followed by identification of the resulting modified oligopeptides showed that cross-link was in C-terminal fragment in positions 131–145, most probably, in decapeptide 131-PGIGATHSSR-140. The position of cross-linking site on the S15 protein did not depend on the nature of the A site-bound codon (sense or stop codon) and on the presence of polypeptide chain release factor eRF1 in the ribosomal complexes with mRNA analogues bearing a stop codon. The results indicate an involvement of the mentioned decapeptide in the formation of the ribosomal decoding site during elongation and termination of translation. Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from eubacteria and archaea, revealed that decapeptide PGIGATHSSR in positions 131–140 is strongly conserved in eukaryotes and has minor variations in archaea but has no homology with any sequence in C-terminal part of eubacterial S19p, which suggests involvement of the decapeptide in the translation process in a eukaryote-specific manner.     

The first position of a codon placed in the A site of the human 80S ribosome contacts nucleotide C1696 of the 18S rRNA as well as proteins S2, S3, S3a, S30, and S15

Messenger RNA analogues (42-mers) containing a GAC codon (aspartic acid) in the middle of their sequence followed by a s(4)UGA stop codon were used to identify the components of the human ribosomal A site in direct contact with the photoactivatable 4-thiouridine (s(4)U) residue. We compared the behavior of the nonphased ribosome-mRNA complex, (-)tRNA(Asp), to the one of the phased complex, (+)tRNA(Asp), in the absence and in the presence of eRF1, the eukaryotic class 1 translation termination factor of human origin. The patterns of cross-links obtained for the three complexes were similar to those previously reported for rabbit ribosomes [Chavatte, L., et al. (2001) Eur. J. Biochem. 268, 2896-2904]. Cross-links involving proteins S2, S3, S3a, and S30 were poorly dependent on the presence of tRNA(Asp) and eRF1. Cross-linking to nucleotide C1696 of 18S rRNA occurred in all complexes, but its yield was at least two times higher in the phased complex with an empty A site than in the nonphased complex or when the A site was occupied by eRF1. In contrast, protein S15 cross-linked only in the phased complex in the absence of eRF1. The data obtained point to notable differences in organization of the decoding site between mammalian and prokaryotic ribosomes and to large internal mobility of the components of the tRNA (eRF1)-free A site.     

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.     

Molecular modeling of positioning of human release factor eRF1 relative to mRNA stop-codon explains a proximity of the eRF1 C-domain to stop-codon in ribosomal complex

A properties of atomic models of structure of eukaryotic triple complex eRF1 . mRNA . tRNAPhe containing human class-1 polypeptide release factor eRF1 at the A-site of human 80S ribosome, mRNA and P-site tRNAPhe, obtained before, are considered. The stricture of the complex is described using high resolution NMR structure of eRF1 M-domain. The structural properties of distribution of chemical cross-links are investigated, which allows us to choose correct model of positioning of the eRF1 molecule in ribosome A-site relative to stop codon of mRNA. A distributions of crosslinks between photoactivatable perfluoroaryl azide group of modified nucleotides of mRNA analogues and eRF1 molecule are modeled via molecular dynamics method. Twelve different mRNA analogues with modified nucleotides of stop signal in positions +4 to +9 with respect to the first nucleotide of the P-site codon are modeled. It was shown that only one of the two models of complex eRFI . mRNA . tRNA gives cross-link distribution in a good agreement with experimental data. A new features of the final structure of triple complex eRF1 . mRNA . tRNA is spatial proximity of stop-codon nucleotides to the C-domain of the eRF1, which explains previously obtained cross-link experimental data.     

Molecular modeling of structure of eukaryotic ribosomal translation termination complex

Models of atomic structure of eukaryotic translation termination complex containing mRNA, P-site tRNAPhe, human class-1 polypeptide release factor eRF1 and 80S ribosome were constructed. The method of computational modeling was applied. The modeling was based on the functional and structural similarity between tRNA and eFR1 bound in the ribosomal A site. Structural template for the modeling was a known structure of the 70S ribosome complexed with mRNA, P- and A-site tRNAsPhe. The eRF1 molecule bound to the ribosome undergone substantial conformational changes resulting in the mutual configuration of the N- and M-domains similar to tRNA shape. Two models of binding of eRF1 to mRNA at the A-site in the presence of P-site tRNA were generated and characterized by a shape complementarity between the mRNA stop codon and grooves of the different sides of the molecular surface of the fragment of alpha2-helix, NIKS loop and alpha-helix of the N-domain. In the model 1 the stop-codon nucleotides were at the equal distances from the N- and C-domains. In the model 2 the stop-codon was proximal to the NIKS and YxCxxxF motifs of the N-domain. Both models fit the genetic and biochemical data available so far.     

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.     

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.     

Model of the structure of the eukaryotic ribosomal translation termination complex

Models of the atomic structure of the eukaryotic translation termination complex containing mRNA, P-site tRNA^Phe, human class 1 release factor eRF1, and 80S ribosome, were constructed by computational modeling. The modeling was based on the assumed structural-functional similarity between the tRNA and eFR1 molecules in the ribosomal A site. The known atomic structure of the 70S ribosome complexed with mRNA as well as the P-and A-site tRNAs^Phe was used as a structural template for the modeling. The eRF1 molecule bound in the A site undergoes substantial conformational changes so that the mutual configuration of the N and M domains matches the overall tRNA shape. Two models of eRF1 binding to mRNA at the A site in the presence of P-site tRNA^Phe were generated. A characteristic of these models is complementary interactions between the mRNA stop codon and the grooves at different sides of the surface of the eRF1 fragment, containing helix α2, NIKS loop, and helix α3 of the N domain. In model 1, the nucleotides of the mRNA stop codon at the A site are approximately equidistant (～15 Å) from the N (motifs NIKS and YxCxxxF) and C domains. In model 2, the stop codon is close to the N-domain motifs NIKS and YxCxxxF. Both models fit genetic and biochemical experimental data. The choice of a particular model requires additional studies.     

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.     

Interplay between termination and translation machinery in eukaryotic selenoprotein synthesis.

Termination of translation in eukaryotes is catalyzed by eRF1, the stop codon recognition factor, and eRF3, an eRF1 and ribosome-dependent GTPase. In selenoprotein mRNAs, UGA codons, which typically specify termination, serve an alternate function as sense codons. Selenocysteine incorporation involves a unique tRNA with an anticodon complementary to UGA, a unique elongation factor specific for this tRNA, and cis-acting secondary structures in selenoprotein mRNAs, termed SECIS elements. To gain insight into the interplay between the selenocysteine insertion and termination machinery, we investigated the effects of overexpressing eRF1 and eRF3, and of altering UGA codon context, on the efficiency of selenoprotein synthesis in a transient transfection system. Overexpression of eRF1 does not increase termination at naturally occurring selenocysteine codons. Surprisingly, selenocysteine incorporation is enhanced. Overexpression of eRF3 did not affect incorporation efficiency. Coexpression of both factors reproduced the effects with eRF1 alone. Finally, we show that the nucleotide context immediately upstream and downstream of the UGA codon significantly affects termination to incorporation ratios and the response to eRF overexpression. Implications for the mechanisms of selenocysteine incorporation and termination are discussed.     

Stop codons and UGG promote efficient binding of the polypeptide release factor eRF1 to the ribosomal A site

To investigate the codon dependence of human eRF1 binding to the mRNA-ribosome complex, we examined the formation of photocrosslinks between ribosomal components and mRNAs bearing a photoactivable 4-thiouridine probe in the first position of the codon located in the A site. Addition of eRF1 to the phased mRNA-ribosome complexes triggers a codon-dependent quenching of crosslink formation. The concentration of eRF1 triggering half quenching ranges from low for the three stop codons, to intermediate for s4UGG and high for other near-cognate triplets. A theoretical analysis of the photochemical processes occurring in a two-state bimolecular model raises a number of stringent conditions, fulfilled by the system studied here, and shows that in any case sound KD values can be extracted if the ratio mT/KD<1 (mT is total concentration of mRNA added). Considering the KD values obtained for the stop, s4UGG and sense codons (approximately 0.06 microM, 0.45 microM and 2.3 microM, respectively) and our previous finding that only the stop and s4UGG codons are able to promote formation of an eRF1-mRNA crosslink, implying a role for the NIKS loop at the tip of the N domain, we propose a two-step model for eRF1 binding to the A site: a codon-independent bimolecular step is followed by an isomerisation step observed solely with stop and s4UGG codons. Full recognition of the stop codons by the N domain of eRF1 triggers a rearrangement of bound eRF1 from an open to a closed conformation, allowing the universally conserved GGQ loop at the tip of the M domain to come into close proximity of the peptidyl transferase center of the ribosome. UGG is expected to behave as a cryptic stop codon, which, owing to imperfect eRF1-codon recognition, does not allow full reorientation of the M domain of eRF1. As far as the physical steps of eRF1 binding to the ribosome are considered, they appear to closely mimic the behaviour of the tRNA/EF-Tu/GTP complex, but clearly eRF1 is endowed with a greater conformational flexibility than tRNA.     

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.     

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.     

Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome

To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31–33, and to lesser extent amino acids within region 121–131 (the “YxCxxxF loop”) in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61–64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of “protein anticodon” type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31–33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.     

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．     

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.     

The effect of eukaryotic release factor depletion on translation termination in human cell lines

Two competing events, termination and readthrough (or nonsense suppression), can occur when a stop codon reaches the A-site of a translating ribosome. Translation termination results in hydrolysis of the final peptidyl-tRNA bond and release of the completed nascent polypeptide. Alternatively, readthrough, in which the stop codon is erroneously decoded by a suppressor or near cognate transfer RNA (tRNA), results in translation past the stop codon and production of a protein with a C-terminal extension. The relative frequency of termination versus readthrough is determined by parameters such as the stop codon nucleotide context, the activities of termination factors and the abundance of suppressor tRNAs. Using a sensitive and versatile readthrough assay in conjunction with RNA interference technology, we assessed the effects of depleting eukaryotic releases factors 1 and 3 (eRF1 and eRF3) on the termination reaction in human cell lines. Consistent with the established role of eRF1 in triggering peptidyl-tRNA hydrolysis, we found that depletion of eRF1 enhances readthrough at all three stop codons in 293 cells and HeLa cells. The role of eRF3 in eukarytotic translation termination is less well understood as its overexpression has been shown to have anti-suppressor effects in yeast but not mammalian systems. We found that depletion of eRF3 has little or no effect on readthrough in 293 cells but does increase readthrough at all three stop codons in HeLa cells. These results support a direct role for eRF3 in translation termination in higher eukaryotes and also highlight the potential for differences in the abundance or activity of termination factors to modulate the balance of termination to readthrough reactions in a cell-type-specific manner.