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1.
Translation termination is catalyzed by release factors that recognize stop codons. However, previous works have shown that in some bacteria, the termination process also involves bases around stop codons. Recently, Ito et al. analyzed release factors and identified the amino acids therein that recognize stop codons. However, the amino acids that recognize bases around stop codons remain unclear. To identify the candidate amino acids that recognize the bases around stop codons, we aligned the protein sequences of the release factors of various bacteria and searched for amino acids that were conserved specifically in the sequence of bacteria that seemed to regulate translation termination by bases around stop codons. As a result, species having several highly conserved residues in RF1 and RF2 showed positive correlations between their codon usage bias and conservation of the bases around the stop codons. In addition, some of the residues were located very close to the SPF motif, which deciphers stop codons. These results suggest that these conserved amino acids enable the release factors to recognize the bases around the stop codons.
Present address (Y. Ozawa): Tokyo Research Laboratory, IBM Japan, Ltd., 1623-14 Shimotsuruma, Yamato-shi, Kanagawa 242-8502, Japan 相似文献
2.
B Vestergaard L B Van G R Andersen J Nyborg R H Buckingham M Kjeldgaard 《Molecular cell》2001,8(6):1375-1382
Bacterial release factor RF2 promotes termination of protein synthesis, specifically recognizing stop codons UAA or UGA. The crystal structure of Escherichia coli RF2 has been determined to a resolution of 1.8 A. RF2 is structurally distinct from its eukaryotic counterpart eRF1. The tripeptide SPF motif, thought to confer RF2 stop codon specificity, and the universally conserved GGQ motif, proposed to be involved with the peptidyl transferase center, are exposed in loops only 23 A apart, and the structure suggests that stop signal recognition is more complex than generally believed. 相似文献
3.
Sarah Matheisl Otto Berninghausen Thomas Becker Roland Beckmann 《Nucleic acids research》2015,43(18):8615-8626
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. 相似文献
4.
5.
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. 相似文献
6.
Liu Q 《Bio Systems》2005,81(3):281-289
Using full-length cDNA sequences, a comparative analysis of sequence patterns around the stop codons in six eukaryotes was performed. Here, it was showed that the codon immediately before and after the stop codons (defined as -1 codon and +1 codon, respectively) were much more biased than other examined positions, especially at the second position of -1 codons and the first position of +1 codons which were rich in As/Us and purines, respectively, for most species. The author speculated that strongly biased sequence pattern from position -2 to +4 might act as an extended translation termination signal. Translation termination was catalyzed by release factors that recognized the stop codons. The multiple amino acid sequence alignment of eukaryotic release factor 1 (eRF1) of 20 species showed that there were 16 residue sites that were strictly conserved, especially the invariant amino acids Ile70 and Lys71. Accordingly, it could be inferred that those candidate amino acids might involve in the recognition process. Moreover, the possible stop signal recognition hypothesis was also discussed herein. 相似文献
7.
Fan-Minogue H Du M Pisarev AV Kallmeyer AK Salas-Marco J Keeling KM Thompson SR Pestova TV Bedwell DM 《Molecular cell》2008,30(5):599-609
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. 相似文献
8.
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 a recognition, making 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
9.
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. 相似文献
10.
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
11.
Identification of eRF1 residues that play critical and complementary roles in stop codon recognition
Conard SE Buckley J Dang M Bedwell GJ Carter RL Khass M Bedwell DM 《RNA (New York, N.Y.)》2012,18(6):1210-1221
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
12.
Nikita Biziaev Elizaveta Sokolova Dmitry V. Yanvarev Ilya Yu Toropygin Alexey Shuvalov Tatiana Egorova Elena Alkalaeva 《The Journal of biological chemistry》2022,298(7)
The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs. 相似文献
13.
Protein synthesis in ribosomes 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. Synthesis is also 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 their anticodons is known. The termination factors are thought to do this. In the large ribosomal RNA, we found two sites that, like tRNAs, contain the anticodon hairpin but with triplets complementary to stop codons. One site is hairpin 69 from domain IV; the other site is hairpin 89, domain V. By analogy, we call them termination tRNAs: Ter-tRNA1 and Ter-tRNA2, respectively, even though they transport no amino acids, and suggest that they directly pair to stop codons. The termination factors only aid in this recognition, making it specific and reliable. A strong argument in favor of our hypothesis comes from vertebrate mitochondria. They are known to acquire two new stop codons, AGA and AGG. In the standard code, these are two out of six arginine codons. We revealed that the corresponding anticodons, UCU and CCU, have evolved in Ter-tRNA1 of these mitochondria. 相似文献
14.
Haritha Vallabhaneni Hua Fan-Minogue David M. Bedwell Philip J. Farabaugh 《RNA (New York, N.Y.)》2009,15(5):889-897
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. 相似文献
15.
The involvement of base 1054 in 16S rRNA for UGA stop codon dependent translational termination. 总被引:6,自引:5,他引:1
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The deletion of the highly conserved cytidine nucleotide at position 1054 in E. coli 16S rRNA has been characterized to confer an UGA stop codon specific suppression activity which suggested a functional participation of small subunit rRNA in translational termination. Based on this structure-function correlation we constructed the three point mutations at site 1054, changing the wild-type C residue to an A, G or U base. The mutations were expressed from a complete plasmid encoded rRNA operon (rrnB) using a conditional expression system with the lambda PL-promoter. All three altered 16S rRNA molecules were expressed and incorporated into 70S ribosomal particles. Structural analysis of the protein and 16S rRNA moieties of the mutant ribosomes showed no differences when compared to wild-type particles. The phenotypic analysis revealed that only the 1054G base change led to a significantly reduced generation time of transformed cells, which could be correlated with the inability of the mutant ribosomes to specifically stop at UGA stop codons in vivo. The response towards UAA and UAG termination codons was not altered. Furthermore, in vitro RF-2 termination factor binding experiments indicated that the association behaviour of mutant ribosomes was not changed, enforcing the view that the UGA stop codon suppression is a direct consequence of the rRNA mutation. Taken together, these results argue for a direct participation of that 16S rRNA motif in UGA dependent translational termination and furthermore, suggest that termination factor binding and stop codon recognition are two separate steps of the termination event. 相似文献
16.
Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1 总被引:2,自引:0,他引:2
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Class-1 polypeptide chain release factors (RFs) play a key role in translation termination. Eukaryotic (eRF1) and archaeal class-1 RFs possess a highly conserved Asn-Ile-Lys-Ser (NIKS) tetrapeptide located at the N-terminal domain of human eRF1. In the three-dimensional structure, NIKS forms a loop between helices. The universal occurrence and exposed nature of this motif provoke the appearance of hypotheses postulating an essential role of this tetrapeptide in stop codon recognition and ribosome binding. To approach this problem experimentally, site-directed mutagenesis of the NIKS (positions 61-64) in human eRF1 and adjacent amino acids has been applied followed by determination of release activity and ribosome-binding capacity of mutants. Substitutions of Asn61 and Ile62 residues of the NIKS cause a decrease in the ability of eRF1 mutants to promote termination reaction in vitro, but to a different extent depending on the stop codon specificity, position, and nature of the substituting residues. This observation points to a possibility that Asn-Ile dipeptide modulates the specific recognition of the stop codons by eRF1. Some replacements at positions 60, 63, and 64 cause a negligible (if any) effect in contrast to what has been deduced from some current hypotheses predicting the structure of the termination codon recognition site in eRF1. Reduction in ribosome binding revealed for Ile62, Ser64, Arg65, and Arg68 mutants argues in favor of the essential role played by the right part of the NIKS loop in interaction with the ribosome, most probably with ribosomal RNA. 相似文献
17.
Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes
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Salas-Marco J Fan-Minogue H Kallmeyer AK Klobutcher LA Farabaugh PJ Bedwell DM 《Molecular and cellular biology》2006,26(2):438-447
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. 相似文献
18.
Gary Loughran Xiang Li Sinead OLoughlin John F Atkins Pavel
V Baranov 《Nucleic acids research》2023,51(1):304
A stop codon entering the ribosome A-site is normally decoded by release factors that induce release of the polypeptide. Certain factors influence the efficiency of the termination which is in competition with elongation in either the same (readthrough) or an alternative (frameshifting) reading frame. To gain insight into the competition between these processes, we monitored translation in parallel from all three reading frames downstream of stop codons while changing the nucleotide context of termination sites or altering cellular conditions (polyamine levels). We found that P-site codon identity can have a major impact on the termination efficiency of the OPRL1 stop signal, whereas for the OAZ1 ORF1 stop signal, the P-site codon mainly influences the reading frame of non-terminating ribosomes. Changes to polyamine levels predominantly influence the termination efficiency of the OAZ1 ORF1 stop signal. In contrast, increasing polyamine levels stimulate readthrough of the OPRL1 stop signal by enhancing near-cognate decoding rather than by decreasing termination efficiency. Thus, by monitoring the four competing processes occurring at stop codons we were able to determine which is the most significantly affected upon perturbation. This approach may be useful for the interrogation of other recoding phenomena where alternative decoding processes compete with standard decoding. 相似文献
19.
Askarian-Amiri ME Pel HJ Guévremont D McCaughan KK Poole ES Sumpter VG Tate WP 《The Journal of biological chemistry》2000,275(23):17241-17248
The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions. 相似文献
20.
The effect of eukaryotic release factor depletion on translation termination in human cell lines 总被引:4,自引:0,他引:4
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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