Termination of translation in bacteria may be modulated via specific interaction between peptide chain release factor 2 and the last peptidyl-tRNA(Ser/Phe).

The 5' context of 671 Escherichia coli stop codons UGA and UAA has been compared with the context of stop-like codons (UAC, UAU and CAA for UAA; UGG, UGC, UGU and CGA for UGA). We have observed highly significant deviations from the expected nucleotide distribution: adenine is over-represented whereas pyrimidines are under-represented in position -2 upstream from UAA. Uridine is over-represented in position -3 upstream from UGA. Lysine codons are preferable immediately prior to UAA. A complete set of codons for serine and the phenylalanine UUC codon are preferable immediately 5' to UGA. This non-random codon distribution before stop codons could be considered as a molecular device for modulation of translation termination. We have found that certain fragment of E. coli release factor 2 (RF2) (amino acids 93-114) is similar to the amino acid sequences of seryl-tRNA synthetase (positions 10-19 and 80-93) and of beta (small) subunit (positions 72-94) of phenylalanyl-tRNA synthetase from E. coli. Three-dimensional structure of E. coli seryl-tRNA synthetase is known [1]: Its N-terminus represents an antiparallel alpha-helical coiled-coil domain and contains a region homologous to RF2. On the basis of the above-mentioned results we assume that a specific interaction between RF2 and the last peptidyl-tRNA(Ser/Phe) occurs during polypeptide chain termination in prokaryotic ribosomes.     

Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes.

An increasing number of cases where tri-nucleotide stop codons do not signal the termination of protein synthesis are being reported. In order to identify what constitutes an efficient stop signal, we analysed the region around natural stop codons in genes from a wide variety of eukaryotic species and gene families. Certain stop codons and nucleotides following stop codons are over-represented, and this pattern is accentuated in highly expressed genes. For example, the preferred signal for Saccharomyces cerevisiae and Drosophila melanogaster highly expressed genes is UAAG, and generally the signals UAA(A/G) and UGA(A/G) are preferred in eukaryotes. The GC% of the organism or DNA region can affect whether there is A or G in the second or fourth positions. We suggest therefore, that the stop codon and the nucleotide following it comprise a tetra-nucleotide stop signal. A model is proposed in which the polypeptide chain release factor, a protein, recognises this sequence, but will tolerate some substitution, particularly A to G in the second or third positions.     

5' contexts of Escherichia coli and human termination codons are similar.

The nearest 5' context of 2559 human stop codons was analysed in comparison with the same context of stop-like codons (UGG, UGC, UGU, CGA for UGA; CAA, UAU, UAC for UAA; and UGG, UAU, UAC, CAG for UAG). The non-random distribution of some nucleotides upstream of the stop codons was observed. For instance, uridine is over-represented in position -3 upstream of UAG. Several codons were shown to be over-represented immediately upstream of the stop codons: UUU(Phe), AGC(Ser), and the Lys and Ala codon families before UGA; AAG(Lys), GCG(Ala), and the Ser and Leu codon families before UAA; and UCA(Ser), AUG(Met), and the Phe codon family before UAG. In contrast, the Thr and Gly codon families were under-represented before UGA, while ACC(Thr) and the Gly codon family were under-represented before UAG and UAA respectively. In an earlier study, uridine was shown to be over-represented in position -3 before UGA in Escherichia coli [Arkov,A.L., Korolev,S.V. and Kisselev,L.L. (1993) Nucleic Acids Res., 21,2891-2897]. In that study, the codons for Lys, Phe and Ser were shown to be over-represented immediately upstream of E. coli stop codons. Consequently, E. coli and human termination codons have similar 5' contexts. The present study suggests that the 5' context of stop codons may modulate the efficiency of peptide chain termination and (or) stop codon readthrough in higher eukaryotes, and that the mechanisms of such a modulation in prokaryotes and higher eukaryotes may be very similar.     

Cis control of gene expression in E.coli by ribosome queuing at an inefficient translational stop signal

An UGA stop codon context which is inefficient because of the 3'-flanking context and the last two amino acids in the gene protein product has a negative effect on gene expression, as shown using a model protein A' gene. This is particularly true at low mRNA levels, corresponding to a high intracellular ribosome/mRNA ratio. The negative effect is smaller if this ratio is decreased, or if the distance between the initiation and termination signals is increased. The results suggest that an inefficient termination codon can cause ribosomal pausing and queuing along the upstream mRNA region, thus blocking translation initiation of short genes. This cis control effect is dependent on the stop codon context, including the C-terminal amino acids in the gene product, the translation initiation signal strength, the ribosome/mRNA ratio and the size of the mRNA coding region. A large proportion of poorly expressed natural Escherichia coli genes are small, and the weak termination codon UGA is under-represented in small, highly expressed E.coli genes as compared with the efficient stop codon UAA.     

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.     

UGA suppression by normal tRNA Trp in Escherichia coli: codon context effects.

The nucleotide sequences at the 3' side of in-phase UGA termination codons in mRNAs of various prokaryotic genes were re-examined. An adenine (A) residue is found to be adjacent to the 3' side of UGA in mRNAs which code for readthrough proteins by the suppression of UGA by normal Escherichia coli tRNA Trp. It is suggested that the nature of the nucleotide following a UGA codon determines whether the UGA signals inefficiently or efficiently the termination of polypeptide chain synthesis: an A residue at this position permits the UGA readthrough process.     

Proline residues at the C terminus of nascent chains induce SsrA tagging during translation termination

The SsrA or tmRNA quality control system relieves ribosome stalling and directs the addition of a degradation tag to the C terminus of the nascent chain. In some instances, SsrA tagging of otherwise full-length proteins occurs when the ribosome pauses at stop codons during normal translation termination. Here, the identities of the C-terminal residues of the nascent chain are shown to play an important role in full-length protein tagging. Specifically, a subset of C-terminal Xaa-Pro sequences caused SsrA tagging of the full-length YbeL protein from Escherichia coli. This tagging increased when a less efficient stop codon was used, increased in cells lacking protein release factor-3, and decreased when protein release factor-1 was overexpressed. Incorporation of the analog azetidine-2-carboxylic acid in place of proline suppressed tagging, whereas incorporation of 3,4-dehydroproline increased SsrA tagging of full-length YbeL. These results suggest that the detailed chemical or conformational properties of the C-terminal residues of the nascent polypeptide can affect the rate of translation termination, thereby influencing ribosome pausing and SsrA tagging at stop codons.     

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.     

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.     

Identification of a novel termination release factor eRF3b expressing the eRF3 activity in vitro and in vivo

Termination of translation in eukaryotes is governed by the ribosome, a termination codon in the mRNA, and two polypeptide chain release factors (eRF1 and eRF3). We have identified a human protein of 628 amino acids, named eRF3b, which is highly homologous to the known human eRF3 henceforth named eRF3a. At the nucleotide and at the amino acid levels the human eRF3a and eRF3b are about 87% identical. The differences in amino acid sequence are concentrated near the amino terminus. The most important difference in the nucleotide sequence is that eRF3b lacks a GGC repeat close to the initiation codon in eRF3a. We have cloned the cDNA encoding the human eRF3b, purified the eRF3b expressed in Escherichia coli, and found that the protein is active in vitro as a potent stimulator of the release factor activity of human eRFl. Like eRF3a, eRF3b exhibits GTPase activity, which is ribosome- and eRFl-dependent. In vivo assays (based on suppression of readthrough induced by three species of suppressor tRNAs: amber, ochre, and opal) show that the human eRF3b is able to enhance the release factor activity of endogenous and overexpressed eRFl with all three stop codons.     

Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.

An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.     

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.     

Comparative study of translation termination sites and release factors (RF1 and RF2) in procaryotes

Translation termination is catalyzed by release factors that recognize stop codons. However, previous works have shown that in some bacteria, the termination process also involves bases around stop codons. Recently, Ito et al. analyzed release factors and identified the amino acids therein that recognize stop codons. However, the amino acids that recognize bases around stop codons remain unclear. To identify the candidate amino acids that recognize the bases around stop codons, we aligned the protein sequences of the release factors of various bacteria and searched for amino acids that were conserved specifically in the sequence of bacteria that seemed to regulate translation termination by bases around stop codons. As a result, species having several highly conserved residues in RF1 and RF2 showed positive correlations between their codon usage bias and conservation of the bases around the stop codons. In addition, some of the residues were located very close to the SPF motif, which deciphers stop codons. These results suggest that these conserved amino acids enable the release factors to recognize the bases around the stop codons. Present address (Y. Ozawa): Tokyo Research Laboratory, IBM Japan, Ltd., 1623-14 Shimotsuruma, Yamato-shi, Kanagawa 242-8502, Japan     

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.     

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.     

Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1-mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from approximately 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.     

Why termination tRNAs have not been found? Because they are hidden in the large ribosomal RNA

It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such a recognition, making it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.     

Why Termination tRNAs Have Not Been Found? Because They Are Hidden in the Large Ribosomal RNA (A Hypothesis)

It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such recognition to make it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.     

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.