Translational termination in Escherichia coli: three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing signals.

The observations that the Escherichia coli release factor 2 (RF2) crosslinks with the base following the stop codon (+4 N), and that the identity of this base strongly influences the decoding efficiency of stop signals, stimulated us to determine whether there was a more extended termination signal for RF2 recognition. Analysis of the 3' contexts of the 1248 genes in the E.coli genome terminating with UGA showed a strong bias for U in the +4 position and a general bias for A and against C in most positions to +10, consistent with the concept of an extended sequence element. Site-directed crosslinking occurred to RF2 from a thio-U sited at the +4, +5 and +6 bases following the UGA stop codon but not beyond (+7 to +10). Varying the +4 to +6 bases modulated the strength of the crosslink from the +1 invariant U to RF2. A strong selection bias for particular bases in the +4 to +6 positions of certain E. coli UGANNN termination sites correlated in some cases with crosslinking efficiency to RF2 and in vivo termination signal strength. These data suggest that RF2 may recognise at least a hexanucleotide UGA-containing sequence and that particular base combinations within this sequence influence termination signal decoding efficiency.     

The translational stop signal: Codon with a context, or extended factor recognition element?

Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal. Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element. A data base of natural termination sites from a wide range of organisms (148 organisms, 40000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4). The nature of this base determines the efficiency of the stop signal in vivo, and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor-3. Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly. Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2. The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context.     

Indirect regulation of translational termination efficiency at highly expressed genes and recoding sites by the factor recycling function of Escherichia coli release factor RF3.

Prokaryotic release factor RF3 is a stimulatory protein that increases the rate of translational termination by the decoding release factors RF1 and RF2. The favoured model for RF3 function is the recycling of RF1 and RF2 after polypeptide release by displacing the factors from the ribosome. In this study, we have demonstrated that RF3 also plays an indirect role in the decoding of stop signals of highly expressed genes and recoding sites by accentuating the influence of the base following the stop codon (+4 base) on termination signal strength. The efficiency of decoding strong stop signals (e.g. UAAU and UAAG) in vivo is markedly improved with increased RF3 activity, while weak signals (UGAC and UAGC) are only modestly affected. However, RF3 is not responsible for the +4 base influence on termination signal strength, since prfC- strains lacking the protein still exhibit the same qualitative effect. The differential effect of RF3 at stop signals can be mimicked by modest overexpression of decoding RF. These findings can be interpreted according to current views of RF3 as a recycling factor, which functions to maintain the concentration of free decoding RF at stop signals, some of which are highly responsive to changes in RF levels.     

The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli.

A statistical analysis of > 2000 Escherichia coli genes suggested that the base following the translational stop codon might be an important feature of the signal for termination. The strengths of each of 12 possible 'four base stop signals' (UAAN, UGAN and UAGN) were tested in an in vivo termination assay that measured termination efficiency by its direct competition with frameshifting. Termination efficiencies varied significantly depending on both the stop codon and the fourth base, ranging from 80 (UAAU) to 7% (UGAC). For both the UAAN and UGAN series, the fourth base hierarchy was U > G > A approximately C. UAG stop codons, which are used rarely in E. coli, showed efficiencies comparable with UAAN and UGAN, but differed in that the hierarchy of the fourth base was G > U approximately A > C. The rate of release factor selection varied 30-fold at UGAN stop signals, and 10-fold for both the UAAN and UAGN series; it correlated well with the frequency with which the different UAAN and UGAN signals are found at natural termination sites. The results suggest that the identity of the base following the stop codon determines the efficiency of translational termination in E. coli. They also provide a rationale for the use of the strong UAAU signal in highly expressed genes and for the occurrence of the weaker UGAC signal at several recording sites.     

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.     

Is the in-frame termination signal of the Escherichia coli release factor-2 frameshift site weakened by a particularly poor context?

The synthesis of release factor-2 (RF-2) in bacteria is regulated by a high efficiency +1 frameshifting event at an in-frame UGA stop codon. The stop codon does not specify the termination of synthesis efficiently because of several upstream stimulators for frameshifting. This study focusses on whether the particular context of the stop codon within the frameshift site of the Escherichia coli RF-2 mRNA contributes to the poor efficiency of termination. The context of UGA in this recoding site is rare at natural termination sites in E.coli genes. We have evaluated how the three nucleotides downstream from the stop codon (+4, +5 and +6 positions) in the native UGACUA sequence affect the competitiveness of the termination codon against the frameshifting event. Changing the C in the +4 position and, separately, the A in the +6 position significantly increase the termination signal strength at the frameshift site, whereas the nucleotide in the +5 position had little influence. The efficiency of particular termination signals as a function of the +4 or +6 nucleotides correlates with how often they occur at natural termination sites in E.coli; strong signals occur more frequently and weak signals are less common.     

Adenine and guanine recognition of stop codon is mediated by different N domain conformations of translation termination factor eRF1

Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125-131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3'-phosphate of UAA, while the same cross-linker at the 3'-phosphate of UAAA modified both the 26-28 and 67-73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines.     

Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast

The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3.     

Release factors and their role as decoding proteins: specificity and fidelity for termination of protein synthesis

The decoding of stop signals in mRNA requires protein release factors. Two classes of factor are found in both prokaryotes and eukaryotes, a decoding factor and a stimulatory recycling factor. These factors form complexes at the active centre of the ribosome and mimic in overall shape the complexes found at other stages of protein synthesis. The decoding release factor is shaped like a tRNA and has a domain for codon recognition at the decoding site of the ribosome, and a domain for peptidyl-tRNA hydrolysis that is inferred to be near the peptidyltransferase centre. Initial interaction of the decoding factor with the ribosome is a low fidelity event involving multiple contacts with the ribosomal components. A subsequent discrimination step, at present poorly defined, ensures high fidelity of codon recognition.     

C-domain of translation termination factor eRF1 neighbors stop codon at the 80S ribosomal A site

Positioning of stop codon and the adjacent triplet downstream of it with respect to the components of human 80S termination complex was studied with the use of mRNA analogues that bore stop signal UPuPuPu (Pu is A or G) and photoactivatable perfluoroaryl azide group. This group was attached to one of nucleotides of the stop signal or 3' of it (in positions +4 to +9 with respect to the first nucleotide of the P site codon). It was shown that upon mild UV irradiation the mRNA analogues crosslinked to components of model complexes imitating state of 80S ribosome in the course of translation termination. It was found that termination factors eRF1 and eRF3 do not affect mutual arrangement of stop signal and the 18S rRNA. Factor eRF1 was shown to cross-link to modified nucleotides in positions +5 to +9 (ability of eRF1 to cross-link to stop codon nucleotide in position +4 was shown earlier). Fragments of eRF1 containing cross-linking sites of the mRNA analogues were determined. In fragment 52-195 (containing the N-domain and a part of the M-domain) we have found cross-linking sites of the analogues that bore modifying groups on A or G in positions +5 to +9 or at the terminal phosphate of nucleotide in position +7. For mRNA analogues bearing modifying groups on G site of cross-linking from positions +5 to +7 was found in the eRF1 fragment     

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

The decoding release factor （RF） triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre （PTC） of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure＂ the anticodon loop and the loop at the tip of helix orS. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.     

Two regions of the Escherichia coli 16S ribosomal RNA are important for decoding stop signals in polypeptide chain termination.

Two regions of the 16S rRNA, helix 34, and the aminoacyl site component of the decoding site at the base of helix 44, have been implicated in decoding of translational stop signals during the termination of protein synthesis. Antibiotics specific for these regions have been tested to see how they discriminate the decoding of UAA, UAG, and UGA by the two polypeptide chain release factors (RF-1 and RF-2). Spectinomycin, which interacts with helix 34, stimulated RF-1 dependent binding to the ribosome and termination. It also stimulated UGA dependent RF-2 termination at micromolar concentrations but inhibited UGA dependent RF-2 binding at higher concentrations. Alterations at position C1192 of helix 34, known to confer spectinomycin resistance, reduced the binding of f[3H]Met-tRNA to the peptidyl-tRNA site. They also impaired termination in vitro, with both factors and all three stop codons, although the effect was greater with RF-2 mediated reactions. These alterations had previously been shown to inhibit EF-G mediated translocation. As perturbations in helix 34 effect both termination and elongation reactions, these results indicate that helix 34 is close to the decoding site on the bacterial ribosome. Several antibiotics, hygromycin, neomycin and tetracycline, specific for the aminoacyl site, were shown to inhibit the binding and function of both RFs in termination with all three stop codons in vitro. These studies indicate that decoding of all stop signals is likely to occur at a similar site on the ribosome to the decoding of sense codons, the aminoacyl site, and are consistent with a location for helix 34 near this site.     

The C domain of translation termination factor eRF1 is close to the stop codon in the A site of the 80S ribosome

The arrangement of the stop codon and its 3′-flanking codon relative to the components of translation termination complexes of human 80S ribosomes was studied using mRNA analogs containing the stop signal UPuPuPu (Pu is A or G) and the photoreactive perfluoroarylazido group, which was linked to a stop-signal or 3′-flanking nucleotide (positions from +4 to +9 relative to the first nucleotide of the P-site codon). Upon mild UV irradiation, the analogs crosslinked to components of the model complexes, mimicking the state of the 80S ribosome at translation termination. Termination factors eRF1 and eRF3 did not change the relative arrangement of the stop signal and 18S rRNA. Crosslinking to eRF1 was observed for modified nucleotides in positions +5 to +9 (that for stop-codon nucleotide +4 was detected earlier). The eRF1 fragments crosslinked to the mRNA analogs were identified. Fragment 52–195, including the N domain and part of the M domain, crosslinked to the analogs carrying the reactive group at A or G in positions +5 to +9 or at the terminal phosphate of nucleotide +7. The site crosslinking to mRNA analogs containing modified G in positions +5 to +7 was assigned to eRF1 fragment 82–166 (beyond the NIKS motif). All but one analog (that with modified G in position +4) crosslinked to the C domain of eRF1 (fragment 330–422). The efficiency of crosslinking to the C domain was higher than to the N domain in most cases. It was assumed that the C domain of eRF1 bound in the A site is close to nucleotides +5 to +9, especially +7 and +8, and that eRF1 undergoes substantial conformational changes when binding to the ribosome.     

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

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

RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites

Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but their low incorporation efficiency, which is possibly due to competition from release factors, limits the power and scope of this technology. Here we show that the reportedly essential release factor 1 (RF1) can be knocked out from Escherichia coli by 'fixing' release factor 2 (RF2). The resultant strain JX33 is stable and independent, and it allows UAG to be reassigned from a stop signal to an amino acid when a UAG-decoding tRNA-synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving new protein functions by enabling Uaa incorporation at multiple sites.     

Comparative analysis of base biases around the stop codons in six eukaryotes

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.     

Rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons

A stop or nonsense codon is an in-frame triplet within a messenger RNA that signals the termination of translation. One common feature shared among all three nonsense codons (UAA, UAG, and UGA) is a uridine present at the first codon position. It has been recently shown that the conversion of this uridine into pseudouridine (Ψ) suppresses translation termination, both in vitro and in vivo. Furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two specific amino acids in a nonsense codon-dependent fashion. Ψ differs from uridine by a single N1H group at the C5 position; how Ψ suppresses termination and, more importantly, enables selective decoding is poorly understood. Here, we provide molecular rationales for how pseudouridylated stop codons are selectively decoded. Our analysis applies crystal structures of ribosomes in varying states of translation to consider weakened interaction of Ψ with release factor; thermodynamic and geometric considerations of the codon-anticodon base pairs to rank and to eliminate mRNA-tRNA pairs; the mechanism of fidelity check of the codon-anticodon pairing by the ribosome to evaluate noncanonical codon-anticodon base pairs and the role of water. We also consider certain tRNA modifications that interfere with the Ψ-coordinated water in the major groove of the codon-anticodon mini-helix. Our analysis of nonsense codons enables prediction of potential decoding properties for Ψ-modified sense codons, such as decoding ΨUU potentially as Cys and Tyr. Our results provide molecular rationale for the remarkable dynamics of ribosome decoding and insights on possible reprogramming of the genetic code using mRNA modifications.     

UGA: a dual signal for 'stop' and for recoding in protein synthesis

UGA remains an enigma as a signal in protein synthesis. Long recognized as a stop signal that is prone to failure when under competition from near cognate events, there was growing belief that there might be functional significance in the production of small amounts of extended proteins. This view has been reinforced with the discovery that UGA is found at some recoding sites where frameshifting occurs as a regulatory mechanism for controlling the gene expression of specific proteins, and it also serves as the code for selenocysteine (Sec), the 21st amino acid. Why does UGA among the stop signals play this role specifically, and how does it escape being used to stop protein synthesis efficiently at recoding sites involving Sec incorporation or shifts to a new translational frame? These issues concerning the UGA stop signals are discussed in this review.     

Escherichia coli release factor 3: resolving the paradox of a typical G protein structure and atypical function with guanine nucleotides.

Escherichia coli release factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction.