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1.
Termination of protein synthesis (hydrolysis of the last peptidyl-tRNA on the ribosome) takes place when the ribosomal A site is occupied simultaneously by one of the three stop codons and by a class-1 translation termination factor. The existing procedures to measure the functional activity of this factor both in vitro and in vivo have serious drawbacks, the main of which are artificial conditions for in vitro assays, far from those in the cell, and indirect evaluation of activity in in vivo systems. A simple reliable and sensitive system to measure the functional activity of class-1 translation termination factors could considerably expedite the study of the terminal steps of protein synthesis, at present remaining poorly known, especially in eukaryotes. We suggest a novel system to test the functional activity in vitro using native functionally active mRNA, rather than tri-, tetra-, or oligonucleotides as before. This mRNA is specially designed to contain one of the three terminating (stop) codons within the coding nucleotide sequence. Plasmids have been generated that carry the genes of suppressor tRNAs each of which is specific toward one of the three stop codons. They were shown to support normal synthesis of a reporter protein, luciferase, by reading through the stop codon within the coding mRNA sequence. We have demonstrated that human class-1 translation termination factor eRF1 is able to compete with suppressor tRNA for a stop codon and to completely prevent its suppressive effect at a sufficient concentration. Forms of eRF1 with point mutations in functionally essential regions have lower competitive ability, demonstrating the sensitivity of the method to the eRF1 structure. The enzymatic reaction catalyzed by the full-size reporter protein is accompanied by emission of light quanta. Therefore, competition between suppressor tRNA and eRF1 can be measured using a luminometer, and this allows precise kinetic measurements in a continuous automatic mode.  相似文献   

2.
Termination of translation in eukaryotes is governed by the ribosome, a termination codon in the mRNA, and two polypeptide chain release factors (eRF1 and eRF3). We have identified a human protein of 628 amino acids, named eRF3b, which is highly homologous to the known human eRF3 henceforth named eRF3a. At the nucleotide and at the amino acid levels the human eRF3a and eRF3b are about 87% identical. The differences in amino acid sequence are concentrated near the amino terminus. The most important difference in the nucleotide sequence is that eRF3b lacks a GGC repeat close to the initiation codon in eRF3a. We have cloned the cDNA encoding the human eRF3b, purified the eRF3b expressed in Escherichia coli, and found that the protein is active in vitroas a potent stimulator of the release factor activity of human eRFl. Like eRF3a, eRF3b exhibits GTPase activity, which is ribosome- and eRFl-dependent. In vivoassays (based on suppression of readthrough induced by three species of suppressor tRNAs: amber, ochre, and opal) show that the human eRF3b is able to enhance the release factor activity of endogenous and overexpressed eRF1 with all three stop codons.  相似文献   

3.
Differential scanning calorimetry (DSC) was used to study thermal denaturation of the human class 1 translation termination factor eRF1 and its mutants. Free energy changes caused by amino acid substitutions in the N domain were computed for eRF1. The melting of eRF1, consisting of three domains, proved to be cooperative. The thermostability of eRF1 was not affected by certain substitutions and was slightly increased by certain others. The corresponding residues were assumed to play no role in maintaining the eRF1 structure, which agreed with the published X-ray data. In these mutants (E55D, Y125F, N61S, E55R, E55A, N61S + S64D, C127A, and S64D), a selective loss of the capability to induce hydrolysis of peptidyl-tRNA in the ribosomal P site in the presence of a stop codon was not associated with destabilization of their spatial structure. Rather, the loss was due to local changes in the stereochemistry of the side groups of the corresponding residues in functionally important sites of the N domain. Two amino acid residues of the N domain, N129 and F131, proved to play an important role in the structural stability of eRF1 and to affect the selective recognition of mRNA stop codons in the ribosome. The recognition of the UAG and UAA stop codons in vitro was more tightly associated with the stability of the spatial structure of eRF1 as compared with that of the UGA stop codon.  相似文献   

4.
The essential eukaryote release factor eRF1, encoded by the yeast SUP45 gene, recognizes stop codons during ribosomal translation. SUP45 nonsense alleles are, however, viable due to the establishment of feedback-regulated readthrough of the premature termination codon; reductions in full-length eRF1 promote tRNA-mediated stop codon readthrough, which, in turn, drives partial production of full-length eRF1. A deterministic mathematical model of this eRF1 feedback loop was developed using a staged increase in model complexity. Model predictions matched the experimental observation that strains carrying the mutant SUQ5 tRNA (a weak UAA suppressor) in combination with any of the tested sup45UAA nonsense alleles exhibit threefold more stop codon readthrough than that of an SUQ5 yeast strain. The model also successfully predicted that eRF1 feedback control in an SUQ5 sup45UAA mutant would resist, but not completely prevent, imposed changes in eRF1 expression. In these experiments, the introduction of a plasmid-borne SUQ5 copy into a sup45UAA SUQ5 mutant directed additional readthrough and full-length eRF1 expression, despite feedback. Secondly, induction of additional sup45UAA mRNA expression in a sup45UAA SUQ5 strain also directed increased full-length eRF1 expression. The autogenous sup45 control mechanism therefore acts not to precisely control eRF1 expression, but rather as a damping mechanism that only partially resists changes in release factor expression level. The validated model predicts that the degree of feedback damping (i.e., control precision) is proportional to eRF1 affinity for the premature stop codon. The validated model represents an important tool to analyze this and other translational negative feedback loops.  相似文献   

5.
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.  相似文献   

6.
The initiation and elongation stages of translation are directed by codon-anticodon interactions. In contrast, a release factor protein mediates stop codon recognition prior to polypeptide chain release. Previous studies have identified specific regions of eukaryotic release factor one (eRF1) that are important for decoding each stop codon. The cavity model for eukaryotic stop codon recognition suggests that three binding pockets/cavities located on the surface of eRF1's domain one are key elements in stop codon recognition. Thus, the model predicts that amino acid changes in or near these cavities should influence termination in a stop codon-dependent manner. Previous studies have suggested that the TASNIKS and YCF motifs within eRF1 domain one play important roles in stop codon recognition. These motifs are highly conserved in standard code organisms that use UAA, UAG, and UGA as stop codons, but are more divergent in variant code organisms that have reassigned a subset of stop codons to sense codons. In the current study, we separately introduced TASNIKS and YCF motifs from six variant code organisms into eRF1 of Saccharomyces cerevisiae to determine their effect on stop codon recognition in vivo. We also examined the consequences of additional changes at residues located between the TASNIKS and YCF motifs. Overall, our results indicate that changes near cavities two and three frequently mediated significant effects on stop codon selectivity. In particular, changes in the YCF motif, rather than the TASNIKS motif, correlated most consistently with variant code stop codon selectivity.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNACys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.  相似文献   

10.
Models of the atomic structure of the eukaryotic translation termination complex containing mRNA, P-site tRNAPhe, 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 tRNAsPhe 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 tRNAPhe 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.  相似文献   

11.
12.
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.  相似文献   

13.
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.  相似文献   

14.
八肋游仆虫两类释放因子的相互作用   总被引:3,自引:1,他引:2  
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。  相似文献   

15.
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.  相似文献   

16.
The iron–sulphur (Fe–S)‐containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in translation termination. Through co‐immunoprecipitation experiments, we show that Rli1 interacts physically with the translation termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35‐21 or sup45‐2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other termination factors. By contrast, RLI1 overexpression partly suppresses the read‐through defects in sup45‐2. Interestingly, we find that although the Fe–S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in translation termination; an Fe–S cluster mutant of RLI1 cannot suppress the read‐through defects of sup45‐2.  相似文献   

17.
In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.  相似文献   

18.
Ivanov  V. I.  Beniaminov  A. D.  Mikheev  A. N.  Minyat  E. E. 《Molecular Biology》2001,35(4):614-622
It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such recognition to make it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

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