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1.
In the translational termination step of protein synthesis the three termination codons UAA, UAG or UGA are recognized by so-called release or termination factors. The release factor RF-1 interacts with UAG and UAA whereas RF-2 is specific for UGA and UAA. Two mechanisms concerning the termination event have been discussed so far: recognition of the termination codon by the protein in a tRNA-like manner or double-strand formation between the codon and the 3' end of the 16S rRNA which is stabilized by the termination factor. Using equilibrium dialysis we show that 40% of the ribosomes can bind UGAA in an RF-2-dependent manner. The stability with the correct combination RF-2-UGA is tenfold higher as compared to the wrong termination codon UAG. We confirm prior findings that the termination factor RF-2 is bound to the A-site of the ribosome. In addition to the ribosomal proteins L2, L10, L7/L12 and L20 of the large subunit and S6 and S18 of the small subunit, the 16S rRNA became labelled when radioactive UGA was crosslinked to the ribosome in the presence of RF-2. Our data support a mechanism of termination in which a double strand between the termination codon and the 3' end of the 16S rRNA is formed as the starting event. The resulting RNA-RNA double strand in turn may be recognized and stabilized by the termination factor.  相似文献   

2.
A single release factor has been isolated and partially purified from rat mitochondria. It requires ethanol in addition to the specific termination codon when assayed in a heterologous system with Escherichia coli ribosomes. The factor recognizes the codons UAA and UAG but not UGA, and therefore it has been designated mtRF-1. A factor of the bacterial RF-2 type, which in E. coli recognizes UGA, or of the mammalian type, which recognizes all three termination codons, has not been detected in mitochondria. The absence of a factor responding to UGA accommodates the use of this codon as a signal for tryptophan in the rat mitochondrial genetic code. The mtRF-1 could translate all of the known termination codons in the rat mitochondrial genome. It does not respond to AGG and AGA which in bovine and human mitochondrial DNA code for termination but which in rat mitochondria may not code for either an amino acid or for termination.  相似文献   

3.
4.
《Gene》1996,169(1):101-103
In Mycoplasma capricolum (Mc), a relative of Gram+ eubacteria with a high genomic A+T-content, the UGA codon is assigned to Trp instead of being a stop codon. We previously showed the lack of peptide-chain release factor (RF) activity in vitro responding to the UGA codon in this bacterium [Inagaki et al., Nucleic Acids Res. 21 (1993) 1335–1338]. To obtain more information on the translation termination mechanism of Mc, we isolated and sequenced the gene encoding RF. The deduced amino-acid sequence has no RF-2-specific + 1 frameshift site and shows 50 and 36% identity to Escherichia coli RF-1 and RF-2, respectively. We conclude that this gene encodes the putative RF-1 which would possess the conserved ‘five-domain’ structure of RF family found in various organisms  相似文献   

5.
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   

6.
Selenoproteins are a family of proteins that share the common feature of containing selenocysteine, the “twenty-first” amino acid. Selenocysteine incorporation occurs during translation of selenoprotein messages by redefinition of UGA codons, which normally specify termination of translation. Studies of the eukaryotic selenocysteine incorporation mechanism suggest that selenocysteine insertion is inefficient compared with termination. Nevertheless, selenoprotein P and several other selenoproteins are known to contain multiple selenocysteines. The production of full-length (FL) protein from these messages would seem to demand highly efficient selenocysteine incorporation due to the compounding effect of termination at each UGA codon. We present data demonstrating that efficient incorporation of multiple selenocysteines can be reconstituted in rabbit reticulocyte lysate translation reactions. Selenocysteine incorporation at the first UGA codon is inefficient but increases by approximately 10-fold at subsequent downstream UGA codons. We found that ribosomes in the “processive” phase of selenocysteine incorporation (i.e., after decoding the first UGA codon as selenocysteine) are fully competent to terminate translation at UAG and UAA codons, that ribosomes become less efficient at selenocysteine incorporation as the distance between UGA codons is increased, and that efficient selenocysteine incorporation is not dependent on cis-acting elements unique to selenoprotein P. Furthermore, we found that the percentage of ribosomes decoding a UGA codon as selenocysteine rather than termination can be increased by 3- to 5-fold by placing the murine leukemia virus UAG read-through element upstream of the first UGA codon or by providing a competing messenger RNA in trans. The mechanisms of selenocysteine incorporation and selenoprotein synthesis are discussed in light of these results.  相似文献   

7.
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   

8.
9.
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   

10.
In none of the 92 published prokaryotic sequences is a translation codon preceeded by UAG as the first "termination codon". In most cases the UAA or UGA is close to the initiation codon and may be part of the ribosome recognition signal.  相似文献   

11.
Recognition of translational termination signals   总被引:4,自引:0,他引:4  
Ribosomes can specifically shift at certain codons so that the mRNA is read in two different reading frames. To determine if frameshifting occurs at the level of termination, polymers of defined sequence containing AUG, a coding sequence and an in- or out-of-phase nonsense codon were used to bind a termination substrate or to program synthesis and release of dipeptides in a highly purified in vitro translation system. fMet-tRNA bound to ribosomes with AUGUAA, AUGUAAn, AUGUUU, AUGUUA or AUGUAn was not a substrate for release factor RF-1. In contrast, AUGU1UAA, AUGU3UAAn, AUGU4UAAn, AUGU5UAAn effected RF-1-dependent release of fMet from ribosomes. This suggests that nonsense codons can stimulate release whether they occur in- or out-of-phase. Addition of exogenous UAA and RF-1 stimulated release with all oligonucleotides tested. Propagation restricted the RF-1-dependent recognition of out-of-phase nonsense codons but did not restrict recognition of in-phase UAA in AUGU3UAAn. Release of dipeptides from ribosomes programmed with AUGU4UAAn occurred without EF-G and with a mutant lacking EF-G activity, suggesting that out-of-phase termination can occur prior to translocation outside the ribosomal A-site. We propose that the ribosome X RF complex is required to complete proteins, but is also able to frameshift at a nonsense codon resulting in occasional out-of-phase termination of protein synthesis.  相似文献   

12.
Bacterial release factors (RFs) 1 and 2 catalyse translation termination at UAG/UAA and UGA/UAA stop codons respectively. It has been shown that limiting the amount of ribosomal protein L11 affects translation termination at UAG and UGA differently. To understand the functional interplay between L11 and RF1/RF2, we isolated 21 distinct mutations in L11 as suppressors of either temperature-sensitive (ts) RF1/RF2 strains or read-through mutants of lacZ nonsense (UAG or UGA) strains. 10 of 21 mutants restored ts lethal growth of RF1 and/or RF2 strains. All the selected L11 mutants, including the RF1ts- and RF2ts-specific suppressors, had the same effect, either enhancing or reducing, on UAG and UGA termination efficiency in vivo. The specific properties of the selected L11 mutations remained unchanged in an RF3 deletion strain. Moreover, ribosomes absent of L11 had equally reduced activity for both RF1- and RF2-mediated peptide release in vitro. These results suggest that, unlike the previous notion, L11 has a common, cooperative role with RF1 and RF2. These L11 mutations were located on the surface of two domains of L11, and interpreted to affect the interaction between L11 and rRNA or the RFs thereby leading to the altered translation termination.  相似文献   

13.
Genomes of mammalian type C retroviruses contain a UAG termination codon between the gag and pol coding regions. The pol region is expressed in the form of a gag-pol fusion protein following readthrough suppression of the UAG codon. We have used oligonucleotide-directed mutagenesis to change the UAG in Moloney murine leukemia virus to UAA or UGA. These alternate termination codons were also suppressed, both in infected cells and in reticulocyte lysates. Thus, the signal or context inducing suppression of UAG in wild-type Moloney murine leukemia virus is also effective with UAA and UGA. Further, mammalian cells and cell extracts contain tRNAs capable of translating UAA and UGA as amino acids. To our knowledge, this is the first example of natural suppression of UAA in higher eucaryotes.  相似文献   

14.
It has been suggested that Escherichia coli release factor 2 (RF-2) translation is autoregulated. Mature RF-2 protein can terminate its own nascent synthesis at an intragenic, in-phase UGA codon, or alternatively, a +1 frameshift can occur that leads to completion of the RF-2 polypeptide. Translational termination presumably increases with RF-2 concentration, providing negative regulatory feedback. We now show, in lacZ/RF-2 fusions, that translation of a UAG codon at the position of the UGA competes with frameshifting, which proves one postulate of the translational autoregulatory model. We also identify a nearby sequence that is required for high-frequency frameshifting and suggest a constraint for the codon preceding the shift point. Both these sequences are incorporated into a model for frameshifting. Our measurements allow us to compute the relative rates in vivo of these reactions: release factor action, frameshifting and tRNA selection at an amber codon.  相似文献   

15.
Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.  相似文献   

16.
The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.  相似文献   

17.
The mechanisms by which polyamines stimulate synthesis of the RNA polymerase sigma(38) subunit in Escherichia coli were studied. Polyamine stimulation was observed only in strains in which the 33rd codon of RpoS mRNA is a UAG termination codon instead of a CAG codon for glutamine in wild-type E. coli. Readthrough of the termination codon by Gln-tRNA(supE) was stimulated by polyamines. This stimulation was found to be caused by an increase in both the level of suppressor tRNA(supE) and the binding affinity of Gln-tRNA(supE) for ribosomes. The stimulatory effect was observed with a UAG termination codon but not with UGA and UAA codons. Readthrough of the UAG termination codon at the 270th amino acid position of RpoS mRNA was also stimulated by polyamines, indicating that polyamines stimulate readthrough of a UAG codon regardless of its location within the RpoS mRNA. When cell viability of an E. coli strain having a termination codon in the 33rd position of RpoS mRNA was compared using cells cultured with or without putrescine, it was higher in cells cultured with putrescine than in cells cultured without putrescine. The level of sigma(38) subunit in the cells cultured with putrescine was higher than that in cells cultured without putrescine on days 2, 4, and 8, but the level of sigma(70) subunit was almost the same in cells cultured with or without putrescine. These results confirm that elevated expression of the rpoS gene is important for cell viability at late stationary phase.  相似文献   

18.
J Heider  C Baron    A Bck 《The EMBO journal》1992,11(10):3759-3766
Incorporation of selenocysteine into proteins is directed by specifically 'programmed' UGA codons. The determinants for recognition of the selenocysteine codon have been investigated by analysing the effect of mutations in fdhF, the gene for formate dehydrogenase H of Escherichia coli, on selenocysteine incorporation. It was found that selenocysteine was also encoded when the UGA codon was replaced by UAA and UAG, provided a proper codon-anticodon interaction was possible with tRNA(Sec). This indicates that none of the three termination codons can function as efficient translational stop signals in that particular mRNA position. The discrimination of the selenocysteine 'sense' codon from a regular stop codon has previously been shown to be dependent on an RNA secondary structure immediately 3' of the UGA codon in the fdhF mRNA. It is demonstrated here that the correct folding of this structure as well as the existence of primary sequence elements located within the loop portion at an appropriate distance to the UGA codon are absolutely required. A recognition sequence can be defined which mediates specific translation of a particular codon inside an mRNA with selenocysteine and a model is proposed in which translation factor SELB interacts with this recognition sequence, thus forming a quaternary complex at the mRNA together with GTP and selenocysteyl-tRNA(Sec).  相似文献   

19.
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   

20.
Effect of base sequence on in vitro protein-chain termination   总被引:4,自引:0,他引:4  
It has been proposed that the sequences surrounding nonsense codons determine the efficiency of protein-chain termination. To test this hypothesis, the termination factor, RF-1, was purified to near homogeneity and was used to examine the specificity of in vitro prokaryotic termination as a function of the nature and number of bases adjacent to UAA. Oligomers with different nucleotide sequences surrounding UAA were synthesized and their conformation was analyzed by NMR spectroscopy. The activity of these oligomers in RF-1-dependent termination was assayed by the release of analogues of peptides, N-acetyl or N-formyl-methionine, that were bound to ribosomes as N-acetyl or N-formyl-Met-tRNAfMet with either AUG or AUG covalently linked to another oligoribonucleotide. In the former case, a second oligomer was added to stimulate release. When added to the AUG-bound intermediate, UAAUAA was 5-fold less effective in stimulating release of N-acetyl-Met by RF-1 than were UAA, UAAN (where N is any base), UAAUGA, or UAAUAG. Oligomers AUGUAA, AUGUUAA, and AUG(U)mUAA18-25 (where m = 1-5) stimulated release by RF-1, whereas AUGCUA, AUGCUAA, and other control polymers were inactive. The data suggest that recognition of UAA depends, at least in part, on the nature of the bases surrounding UAA. A loosely stacked conformation of UAA in the short messengers favors termination, whereas nucleosides which encourage strong base stacking restrict release.  相似文献   

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