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.     

An efficient translational termination of human erythropoietin in Escherichia coli by altering the base following the stop codon

Each of the four nucleotides (A, C, G and T) was introduced as the base following the stop codon to investigate the effect of this fourth base on translational termination efficiency during the heterologous expression of human erythropoietin (hEPO) in E. coli. The efficiency of peptide chain termination in E. coli was markedly dependent on the fourth base. The choice of the fourth base was crucial to prevent the expression of undesirable proteins due to the translational infidelity such as frameshifting and stop codon read-through, and translational termination efficiency could be improved with adenosine as the fourth base.     

Impact of the six nucleotides downstream of the stop codon on translation termination

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3′ termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence –CA(A/G)N(U/C/G)A–, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3′ stop codon context on translation termination.     

Decoding the translational termination signal: the polypeptide chain release factor in Escherichia coli crosslinks to the base following the stop codon.

Protein release factors act like tRNA analogues in decoding translational stop signals. Statistical analysis of the sequences at translational stop sites and functional studies with particular signals indicate this mimicry involves an increase in the length of the signal in the mRNA. The base following the stop codon (+4 base) is of particular interest because it has a strong influence on the competitiveness of the stop signal at recoding sites, suggesting it might form part of the release factor recognition element. Site-directed crosslinking from the +4 base showed that it is in close proximity to the Escherichia coli release factor-2 in a termination complex, a prerequisite for the +4 base being part of the recognition element. Fingerprinting analysis indicates that crosslinking to the release factor occurred from both +1 and +4 positions of the stop signal in the same RNA molecule. This provides more evidence that the +4 base may be an integral part of the decoding signature in the mRNA during the termination phase of protein biosynthesis.     

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.     

TransTerm: a database of translational signals.

The TransTerm database of sequence contexts of stop and start codons has been expanded to include approximately 50% more species than last year's release. It now contains 148 organisms and >39 500 coding sequences; it is now available on the World Wide Web. The database includes: (i) initiation and termination sequence contexts organized by species; (ii) summary parameters about the individual sequences (sequence length, GC%, GC3, Nc, CAI) in addition to tables of base frequencies for each species' stop and start codon sequence context; (iii) species codon usage tables; and (iv) summary tables of stop signal frequency.     

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.     

The major 5' determinant in stop codon read-through involves two adjacent adenines

The aim of this approach was to identify the major determinants, located at the 5′ end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae. We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene. Variations at these positions modulated translational read-through efficiency ~16-fold. The major effect was imposed by the two nucleotides immediately upstream of the stop codon. We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the ribosomal P site. We propose that the mRNA structure, depending on the nucleotides in the P site, is the main 5′ determinant of read-through efficiency.     

The influence of base identity and base pairing on the function of the alpha-sarcin loop of 23S rRNA.

The alpha-sarcin loop of large subunit rRNAs is one of the sites of interaction of elongation factors with the ribosome, and the target of the cytotoxins alpha-sarcin and ricin. Using a genetic selection for increased frameshifting in a reporter gene, we have isolated a C --> U mutation at position 2666 in the alpha-sarcin loop. In the NMR-derived structure of the loop, bases equivalent to 2666 and 2654 are paired via a non-canonical base pairing interaction. Each of the three base substitutions at C2666 and A2654 was constructed by site-directed mutagenesis of a plasmid borne copy of the rrnB operon of Escherichia coli. Only the C2666 --> U and A2654 --> G mutations that resulted in the formation of canonical A-U and C-G base pairs respectively, increased the levels of stop codon readthrough and frameshifting. The effects of different base pair combinations at positions 2666 and 2654 on ribosome function were then tested by constructing and analyzing all possible base combinations at these sites. All A --> G base substitution mutations at position 2654 and C --> U substitutions at position 2666 increased the levels of translational errors. However, these effects were greatest when G2654 and U2666 had the potential to engage in standard Watson-Crick base pairing interactions. These data indicate that base identity as well as base pairing interactions are important for the function of this essential component of the large subunit rRNA.     

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.     

Comparison of characteristics and function of translation termination signals between and within prokaryotic and eukaryotic organisms

Six diverse prokaryotic and five eukaryotic genomes were compared to deduce whether the protein synthesis termination signal has common determinants within and across both kingdoms. Four of the six prokaryotic and all of the eukaryotic genomes investigated demonstrated a similar pattern of nucleotide bias both 5′ and 3′ of the stop codon. A preferred core signal of 4 nt was evident, encompassing the stop codon and the following nucleotide. Codons decoded by hyper-modified tRNAs were over-represented in the region 5′ to the stop codon in genes from both kingdoms. The origin of the 3′ bias was more variable particularly among the prokaryotic organisms. In both kingdoms, genes with the highest expression index exhibited a strong bias but genes with the lowest expression showed none. Absence of bias in parasitic prokaryotes may reflect an absence of pressure to evolve more efficient translation. Experiments were undertaken to determine if a correlation existed between bias in signal abundance and termination efficiency. In Escherichia coli signal abundance correlated with termination efficiency for UAA and UGA stop codons, but not in mammalian cells. Termination signals that were highly inefficient could be made more efficient by increasing the concentration of the cognate decoding release factor.     

Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae

In-frame stop codons normally signal termination during mRNA translation, but they can be read as ‘sense’ (readthrough) depending on their context, comprising the 6 nt preceding and following the stop codon. To identify novel contexts directing readthrough, under-represented 5′ and 3′ stop codon contexts from Saccharomyces cerevisiae were identified by genome-wide survey in silico. In contrast with the nucleotide bias 3′ of the stop codon, codon bias in the two codon positions 5′ of the termination codon showed no correlation with known effects on stop codon readthrough. However, individually, poor 5′ and 3′ context elements were equally as effective in promoting stop codon readthrough in vivo, readthrough which in both cases responded identically to changes in release factor concentration. A novel method analysing specific nucleotide combinations in the 3′ context region revealed positions +1,2,3,5 and +1,2,3,6 after the stop codon were most predictive of termination efficiency. Downstream of yeast open reading frames (ORFs), further in-frame stop codons were significantly over-represented at the +1, +2 and +3 codon positions after the ORF, acting to limit readthrough. Thus selection against stop codon readthrough is a dominant force acting on 3′, but not on 5′, nucleotides, with detectable selection on nucleotides as far downstream as +6 nucleotides. The approaches described can be employed to define potential readthrough contexts for any genome.     

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.     

Influence of the three nucleotides upstream of the initiation codon on expression of the Escherichia coli lacZ gene in Saccharomyces cerevisiae.

By introducing synthetic oligonucleotides into a lacZ-yeast expression vector a set of 47 plasmids (out of 64 possible) was generated, differing only in the three bases immediately upstream of the AUG initiation codon of the Escherichia coli lacZ gene. Expression of the beta-galactosidase fusion protein encoded by the different plasmids was determined in Saccharomyces cerevisiae by immunogel electrophoresis. Among the clones tested we found a factor 3 difference in expression. A slight nucleotide preference was found in positions -3(A > G > C = U) and -2 (G > C = U > A). The choice of the nucleotide at position -1 immediately 5' of the AUG did not effect translation efficiency. Increasing homology to the yeast consensus sequence (AAAAAAAUGUCU) was not concomitant with an increased translation efficiency. Our results indicate that the choice of nucleotides immediately preceding the initiation codon in yeast does not dramatically influence translation efficiency, as in prokaryotes or higher eukaryotes.     

The signal for the termination of protein synthesis in procaryotes.

The sequences around the stop codons of 862 Escherichia coli genes have been analysed to identify any additional features which contribute to the signal for the termination of protein synthesis. Highly significant deviations from the expected nucleotide distribution were observed, both before and after the stop codon. Immediately prior to UAA stop codons in E. coli there is a preference for codons of the form NAR (any base, adenine, purine), and in particular those that code for glutamine or the basic amino acids. In contrast, codons for threonine or branched nonpolar amino acids were under-represented. Uridine was over-represented in the nucleotide position immediately following all three stop codons, whereas adenine and cytosine were under-represented. This pattern is accentuated in highly expressed genes, but is not as marked in either lowly expressed genes or those that terminate in UAG, the codon specifically recognised by polypeptide chain release factor-1. These observations suggest that for the efficient termination of protein synthesis in E. coli, the 'stop signal' may be a tetranucleotide, rather than simply a tri-nucleotide codon, and that polypeptide chain release factor-2 recognises this extended signal. The sequence following stop codons was analysed in genes from several other procaryotes and bacteriophages. Salmonella typhimurium, Bacillus subtilis, bacteriophages and the methanogenic archaebacteria showed a similar bias to E. coli.     

A rRNA-mRNA base pairing model for UGA-dependent termination.

A series of site-directed mutations has been constructed in E coli 16S rRNA and shown to suppress UGA-dependent translational termination. With the exception of the C726 to G base change, all were constructed in helix 34. Characterization of these mutations is reviewed here and from these data and mRNA-rRNA base pairing model for the termination event is presented. The interaction functions via antiparallel base pairing between either 1 of the 2 UCA motifs in helix 34 and the complementary UGA stop codon on the message, thus forming a quasicontinuous A-type helical structure that is further stabilized by stacking enthalpy. Finally, rRNA motifs potentially required for UAA and UAG-dependent translational termination are discussed.     

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal,ochre, and amber suppressor tRNAs

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

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.     

Functional correction of CFTR mutations in human airway epithelial cells using adenine base editors

Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38–82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.