Frameshifting at the internal stop codon within the mRNA for bacterial release factor-2 on eukaryotic ribosomes

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. High-efficiency frameshifting around this codon can occur on eukaryotic ribosomes as well as prokaryotic ribosomes. This was determined from the relative efficiency of translation of RF-2 RNA compared with that for the other release factor RF-1, which lacks the in-frame premature stop codon. Since the termination product is unstable an absolute measure of the efficiency of frameshifting has not been possible. A gene fusion between trpE and RF-2 was carried out to give a stable termination product as well as the frameshift product, thereby allowing a direct determination of frameshifting efficiency. The extension of RF-2 RNA near its start codon with a fragment of the trpE gene, while still allowing high efficiency frameshifting on prokaryotic ribosomes, surprisingly gives a different estimate of frameshifting on the eukaryotic ribosomes than that obtained with RF-2 RNA alone. This paradox may be explained by long distance context effects on translation rates in the frameshift region created by the trpE sequences in the gene fusion, and may reflect that pausing and translation rate are fundamental factors in determining the efficiency of frameshifting.     

Codon recognition in polypeptide chain termination: site directed crosslinking of termination codon to Escherichia coli release factor 2.

An RNA synthesized in vitro was positioned on the Escherichia coli ribosome at the P site with tRNAala, and with a termination codon, UAA, as the next codon in the A site. Such a complex bound stoichiometric amounts of release factor 2 (RF-2); a corresponding RNA with UAC in place of UAA was not a template for the factor. An RNA containing 4-thio-UAA in place of the UAA supported binding of RF-2, and this has allowed site-directed crosslinking from the first position of the termination codon to answer two long standing questions about the termination of protein biosynthesis, the position of the termination codon and its proximity to the release factor during codon recognition. An RF-2.mRNA crosslinked product was detected, indicating the release factor and the termination codon are in close physical contact during the codon recognition event of termination. The 4-thio-U crosslinked also to the ribosome but only to the 30S subunit, and the proteins and the rRNA site concerned were identified. RF-2 decreased significantly the crosslinking to the ribosomal components, but no new crosslink sites were found. If the stop codon was deliberately displaced from the decoding site by one codon's length then a different pattern of crosslinking in particular to the rRNA resulted. These observations are consistent with a model of codon recognition by RF-2 at the decoding site, without a major shift in position of the codon.     

Recognition of translational termination signals

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.     

On the role of the termination factor RF-2 and the 16S RNA in protein synthesis

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.     

The Escherichia coli ribosomal protein L11 suppresses release factor 2 but promotes the release factor 1 activities in peptide chain termination

The incubation of the 50 S ribosomal subunits of Escherichia coli with 1.5 M LiCl yields 1.5c core particles depleted in 14 proteins and inactive in peptide chain termination. In codon-dependent peptidyl-tRNA hydrolysis the release factor 1 (RF-1)-induced reaction essentially depends on both L11 and L16 whereas the release factor 2 (RF-2)-induced reaction is depressed by L11 and stimulated by L16. Omission of L11 results in a several-fold increase in the specific activity of the RF-2. Functional complexes are formed with RF-2 at an apparent Km (dissociation constant) for the termination codon 5-fold lower than with reconstituted ribosomes containing L11; the Vmax for the hydrolysis is unchanged. L11 suppresses this effect when added to the core at close to molar equivalence. In contrast, RF-1 has a very low activity if ribosomes lack L11 and this can be restored by titration of L11 back to the core. This is the first example of a differential or an opposite effect of a ribosomal component on the activities of the two release factors, and the studies suggest that L11 has a critical role in the binding domain for the two factors.     

肽链释放因子识别终止密码子的机制

肽链释放因子(polypeptide release factor, RF)是参与细胞内蛋白质合成终止过程中新生肽链释放的一组重要的蛋白质,包括两类,即第一类肽链释放因子(classⅠrelease factor, RFⅠ)和第二类肽链释放因子(classⅡrelease factor, RFⅡ).关于第一类肽链释放因子识别终止密码子的机制和功能位点是目前分子细胞生物学领域的一个研究热点,第二类肽链释放因子作为一类GTP酶,在第一类肽链释放因子识别终止密码子和肽链释放过程中的协同作用也备受关注.近些年来,通过构建体内和体外的测活体系,对第一类肽链释放因子识别终止密码子的机制的研究取得了一些进展,提出了多种假说和模型,尤其是对第一类肽链释放因子的晶体结构及两类肽链释放因子复合体的空间结构的研究,为揭示真核生物细胞内蛋白质合成终止机制提供了直接的证据.     

Lack of peptide-release activity responding to codon UGA in Mycoplasma capricolum.

In Mycoplasma capricolum, a relative of Gram-positive eubacteria with a high genomic AT-content (75%), codon UGA is assigned to tryptophan instead of termination signal. Thus, in this bacterium the release factor 2 (RF-2), that recognizes UAA and UGA termination codons in eubacteria such as Escherichia coli and Bacillus subtilis, would be either specific to UAA or deleted. To test this, we have constructed a cell-free translation system using synthetic mRNA including codon UAA [mRNA(UAA)], UAG [mRNA(UAG)] and UGA [mRNA(UGA)] in-frame. In the absence of tryptophan, the translation of mRNA(UGA) ceased at UGA sites without appreciable release of the synthesized peptides from the ribosomes, whereas with mRNA(UAA) or mRNA(UAG) the bulk of the peptides was released. Upon addition of the E.coli S-100 fraction or B.subtilis S-100 fraction to the translation system, the synthesized peptides with mRNA(UGA) were almost completely released from the ribosomes, presumably because of the presence of RF-2 active to UGA in the added S-100 fraction. These data suggest that RF-2 is deleted or its activity to UGA is strongly weakened in M.capricolum.     

Isolation of a rat mitochondrial release factor. Accommodation of the changed genetic code for termination

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.     

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.     

Requirements for in vitro reconstruction of protein synthesis

Translation of f2am3 RNA and MS2 RNA in a system containing purified ribosomes and precharged aminoacyl-tRNA, does not occur in the presence of the "known" initiation (IF-1, IF-2 and IF-3), elongation (EF-Tu, EF-Ts and EF-G) and termination (RF-1, RF-2) factors. Translation in this system requires at least three additional protein factors. These are EF-P and the rescue protein as well as a previously undescribed factor we call W, which is found in the high-salt ribosomal eluate.     

The ribosomal binding domain of the Escherichia coli release factors. Modification of tyrosine in the N-terminal domain of ribosomal protein L11 affects release factors 1 and 2 differentially

Ribosomal protein L11 is one of only two ribosomal proteins significantly iodinated when Escherichia coli 50 S subunits are modified by immobilized lactoperoxidase, and the major target has been shown previously to be tyrosine at position 7 in the N-terminal domain. This modification reduces in vitro termination activity with release factor (RF)-1 by 70-90%, but RF-2 activity is less affected (30-50%). The loss of activity parallels incorporation of iodine into the subunit. The 50 S subunits from L11-lacking strains of bacteria have highly elevated activity with RF-2 and low activity with RF-1. The iodination does not affect RF-2 activity but reduces the RF-1 activity further. Ribosomal proteins, L2, L6, and L25, are significantly labeled in L11-lacking ribosomes in contrast to the control 50 S subunits. L11 has been modified in isolation and incorporated back efficiently into L11-lacking ribosomes. This L11, iodinated also predominantly at Tyr 7, is unable to restore RF-1 activity to L11-lacking ribosomes in contrast to mock-iodinated protein. These results suggest the involvement of the N terminus of L11 in the binding domain of the bacterial release factors and indicate that there are subtle differences in how the two factors interact with the ribosome.     

Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2

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.     

The involvement of base 1054 in 16S rRNA for UGA stop codon dependent translational termination.

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.     

Effect of base sequence on in vitro protein-chain termination

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.     

Role of ribosome recycling factor (RRF) in translational coupling

RNA phage GA coat and lysis protein expression are translationally coupled through an overlapping termination and initiation codon UAAUG. Essential for this coupling are the proximity of the termination codon of the upstream coat gene to the initiation codon of the lysis gene (either a <3 nucleotide separation or physical closeness through a possible hairpin structure) but not the Shine-Dalgarno sequence. This suggests that the ribosomes completing the coat gene translation are exclusively responsible for translation of the lysis gene. Inactivation of ribosome recycling factor (RRF), which normally releases ribosomes at the termination codon, did not influence the expression of the reporter gene fused to the lysis gene. This suggests the possibility that RRF may not release ribosomes from the junction UAAUG. However, RRF is essential for correct ribosomal recognition of the AUG codon as the initiation site for the lysis gene.     

蛋白质生物合成的第四步：翻译终止后复合物的解体

蛋白质合成过程一般被归纳为由合成的起始、肽链的延伸和合成的终止组成的三步曲 . 然而,随着对核糖体再循环因子 (ribosome recycling factor , RRF) 在蛋白质合成过程中作用的深入研究,人们提出了蛋白质生物合成应是四步曲, 这第四步就是翻译终止后核糖体复合物的解体 , 也就是通常说的核糖体循环再利用 . 简要地介绍了翻译终止后复合物解体的可能机制：核糖体再循环因子和蛋白质合成延伸因子 G 在核糖体上协同作用催化这一过程的完成 .     

Modification of Escherichia coli ribosomes: in vitro termination is less dependent on histidine residues at the peptidyl transferase centre when ribosomes lack protein L11

Chemical modification of ribosomes with the histidine specific reagents, 1-fluoro-2,4-dinitrobenzene (FDNB) and diethylpyrocarbonate (DEP), result in a loss of activities in vitro of codon-dependent termination and peptide bond formation. The binding of release factor (RF) to the ribosome is unaffected but the hydrolysis of peptidyl-tRNA is inhibited. On reversal of the modification activity can be restored. Partial protection is provided by chloramphenicol indicating that one or more of the affected residues is at the peptidyl transferase centre. Codon-dependent termination on ribosomes lacking L11, which have a greater affinity for RF-2, is significantly less affected by the modification than on control ribosomes. Peptide bond formation is affected similarly on L11 lacking and normal ribosomes.     

Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex.

RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets. We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.     

Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.

Termination of translation in higher organisms is a GTP-dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.