Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro.

Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.     

Glutamine is incorporated at the nonsense codons UAG and UAA in a suppressor-free Escherichia coli strain

Readthrough of the nonsense codons UAG, UAA, and UGA is seen in Escherichia coli strains lacking tRNA suppressors. Earlier results indicate that UGA is miscoded by tRNA(Trp). It has also been shown that tRNA(Tyr) and tRNA(Gln) are involved in UAG and UAA decoding in several eukaryotic viruses as well as in yeast. Here we have investigated which amino acid(s) is inserted in response to the nonsense codons UAG and UAA in E. coli. To do this, the stop codon in question was introduced into the staphylococcal protein A gene. Protein A binds to IgG, which facilitates purification of the readthrough product. We have shown that the stop codons UAG and UAA direct insertion of glutamine, indicating that tRNA(Gln) can read the two codons. We have also confirmed that tryptophan is inserted in response to UGA, suggesting that it is read by tRNA(Trp).     

UAG readthrough is not increased in vivo by Moloney murine leukemia virus infection.

Expression of the pol gene of the murine leukemia viruses is subject to translational control at the UAG termination codon of the upstream gene gag. Previous experiments have suggested that: i) Moloney murine leukemia virus infection induces a tRNA(Gln)iii) in an in vitro system using the tobacco mosaic virus as template, this tRNA is able to increase readthrough at the UAG codon [1]. Here we demonstrate that, in vivo, Moloney murine leukemia virus infection does not increase translational readthrough at either the tobacco mosaic virus or the Moloney murine leukemia virus UAG stop codons.     

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.     

Plant tRNA suppressors: In vivo readthrough properties and nucleotide sequence of yellow lupin seeds tRNA

Transfer RNAs isolated from yellow lupin seeds were screened for tRNA dependent nonsense suppression in Xenopus laevis oocyte system. No opal (UGA) suppressor activity could be detected. However, a tRNA^Tyr isoacceptor species stimulates readthrough of the leaky UAG tobacco mosaic virus (TMV)-RNA stop codon. This natural suppressor tRNA was purified to homogeneity and its sequence was determined to be: pC---C---g--- A---C---C---U---U---A---m²G---c---U---C---A---G---D---G---Gm--- G---U---A---G---A---G---Cm₂²G---G---A---G---G---A---C---U---G--- ψ---A---m¹G---A---ψ---C---C---U---U---A---G---m⁷G---acp³U---C--- A---C---U---G---G---U---ψ---C---G---m¹A---A---Uz.sbnd;C---C---G--- G---U---A---G---G---U---C---G---G---A---C---C---A_OH. The GψA anticodon seqeunce, the presence of the modified nucleoside 3-(3-amino-3-carboxypropyl)uridine (acp³U) in the extra loo p and the absence of ribothymidine are of special interest.     

tRNA hopping: enhancement by an expanded anticodon.

At a low level wild-type tRNA(1Val) inserts a single amino acid (valine) for the five nucleotide sequence GUGUA which has overlapping valine codons. Mutants of tRNA(1Val) with an insertion of A or U between positions 34 and 35 of their anticodons have enhanced reading of the quintuplet sequences. We propose that this decoding occurs by a hopping mechanism rather than by quintuplet pairing. Such hopping involves disengagement of the paired codon and anticodon with the mRNA slipping two (or more) bases along the ribosomal--peptidyl tRNA complex and subsequently re-pairing at a second codon--the landing site. The mutant with the anticodon sequence 3'CAAU5' 'hops' over the stop codon in the mRNA sequence GUG UAA GUU with the insertion of a single amino acid (valine). In contrast, in reading the same sequence, the mutant with the anticodon 3'CAUU5' hops onto the stop with the insertion of two valine residues. It is likely that in some instances of hopping alternate anticodon bases are used for the initial pairing and at the landing site.     

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNA^Gln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.     

Analysis of leaky viral translation termination codons in vivo by transient expression of improved β-glucuronidase vectors

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of -glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.     

Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants

Background

Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain “stop” codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a “readthrough”, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context.

Methodology

We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP.

Conclusions

We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a ‘leaky’ UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context.     

Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs

Translational stop codon readthrough provides a regulatory mechanism of gene expression that is extensively utilised by positive-sense ssRNA viruses. The misreading of termination codons is achieved by a variety of naturally occurring suppressor tRNAs whose structure and function is the subject of this survey. All of the nonsense suppressors characterised to date (with the exception of selenocysteine tRNA) are normal cellular tRNAs that are primarily needed for reading their cognate sense codons. As a consequence, recognition of stop codons by natural suppressor tRNAs necessitates unconventional base pairings in anticodon–codon interactions. A number of intrinsic features of the suppressor tRNA contributes to the ability to read non-cognate codons. Apart from anticodon–codon affinity, the extent of base modifications within or 3′ of the anticodon may up- or down-regulate the efficiency of suppression. In order to out-compete the polypeptide chain release factor an absolute prerequisite for the action of natural suppressor tRNAs is a suitable nucleotide context, preferentially at the 3′ side of the suppressed stop codon. Three major types of viral readthrough sites, based on similar sequences neighbouring the leaky stop codon, can be defined. It is discussed that not only RNA viruses, but also the eukaryotic host organism might gain some profit from cellular suppressor tRNAs.     

The primary structure of six leucine isoacceptor tRNAs of yellow lupin seeds. The structural requirements for amber tRNA suppressor activity

Six tRNA(Leu) isoacceptors from yellow lupin seeds were purified, sequenced, and their readthrough properties over the UAG stop codon were tested using TMV RNA as a messenger. The tested tRNAs(Leu) did not show amber suppressor activity. The partial structure of tRNA(Gln), a minor species in yellow lupin, was also determined. Comparison of the nucleotide sequence of all known isoacceptors of tRNA(Tyr), tRNA(Gln) and tRNA(Leu) from plants, mammals and ciliates enabled us to find general structural requirements for tRNA to be a UAG suppressor. From the partial sequence of lupin tRNA(Gln) we suggest that it will have readthrough properties.     

Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein.

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

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.     

Effects of the nucleotide 3' to an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1

Rates of ribosomal selection of both release factor 1 (RF1) and a suppressor tRNA (Su7C33) were studied at an amber codon at which the 3' neighbor was permuted. Rates of RF1 selection vary 2.6-fold among contexts. The 3' neighbor-dependent variation of RF1 action correlates very strongly with the non-random frequencies of 3' neighbors at UAG terminators (r = 0.97), which argues that the rate of RF1 selection is an important determinant 3' neighbor choice at termination codons. The data are consistent with a model for RF1 selection in which RF1 makes a specific contact(s) to the 3' neighbor and that this interaction is most favorable to uridylic acid. Measured rates of Su7C33 selection vary fivefold among 3' contexts. We also develop a method to calculate rates of selection for other suppressors, based on the assumption that rates of RF1 selection at each 3' context can be generalized to other sites that have the same 3' neighbor. Rates for various suppressors appear to vary from two- to fivefold depending on the 3' neighbor. Generally, the rate of selection of suppressors at different contexts correlates with the stacking strength of the 3' neighbor as measured in vitro. The two- to fivefold range of 3' neighbor effects on rate of aminoacyl-tRNA selection is greater than that previously observed within sets of codons read by the same tRNA. It is suggested that the choice of codons to achieve favorable contexts may be more important than the choice of a common codon at some message sites.     

The leaky UGA termination codon of tobacco rattle virus RNA is suppressed by tobacco chloroplast and cytoplasmic tRNAs(Trp) with CmCA anticodon.

RNA-1 molecules from tobacco rattle virus (TRV) and pea early-browning virus (PEBV), two members of the tobravirus group, have recently been shown to contain internal, in-frame UGA termination codons which are suppressed in vitro. Our results suggest that a UGA stop codon also exists in RNA-1 of pepper ringspot virus (PRV), another tobravirus. UGA suppression may therefore be a universal feature of the expression of tobravirus genomes. We have isolated two natural suppressor tRNAs from uninfected tobacco plants on the basis of their ability to promote readthrough over the leaky UGA codon of TRV RNA-1 in a wheat germ extract depleted of endogenous mRNAs and tRNAs. Their amino acid acceptance and nucleotide sequences identify the two UGA-suppressor tRNAs as chloroplast (chl) and cytoplasmic (cyt) tryptophan-specific tRNAs with the anticodon CmCA. These are the first UGA suppressor tRNAs to be identified in plants. They have several interesting features. (i) Chl tRNA(Trp) suppresses the UGA stop codon more efficiently than cyt tRNA(Trp). (ii) Chl tRNA(Trp) contains an A24:U11 pair in the D-stem as does the mutated Escherichia coli UGA-suppressor tRNA(Trp) which is a more active suppressor than wild-type tRNA(Trp). (iii) The suppressor activity of chl tRNA(Trp) is dependent on the nucleotides surrounding the stop codon because it recognizes UGA in the TRV context but not the UGA in the beta-globin context.     

Exceptional codon recognition by the glutamine tRNAs in Saccharomyces cerevisiae.

Recently, it was shown that wild-type glutamine tRNAs in yeast cause low-level nonsense suppression that can be enhanced by increasing glutamine tRNA gene copy number. In order to investigate glutamine tRNA behavior further, anticodon mutations that confer nonsense suppression were identified in yeast sup70 gene, which codes for glutamine tRNA(CAG). In this study we show that suppressors derived by mutation severely limit growth such that suppressor-bearing spores germinate but arrest cell division at approximately the 50 cell stage. Analysis of a sup70 deletion was used to establish that growth limitation results from loss of wild-type glutamine tRNA(CAG) function. By exploiting the growth inhibition of sup70 alleles, some exceptional codon recognition properties of glutamine tRNAs were revealed. Our results indicate that amber suppressor glutamine tRNA(UAG) can translate 5'-CAG-3' glutamine codons with low efficiency in the presence of an A/C mismatch at the first position of the codon, suggesting that reading may occur at a low level by a two-out-of-three reading mechanism. In addition, when glutamine tRNA(CAA) is over-expressed in vivo, it translates 5'-CAG-3' codons using a mechanism that resembles prokaryotic-like U/G wobble, which normally does not occur in yeast. Our studies also suggest that the yeast glutamine tRNA suppressors could potentially be exploited to express ciliated protozoan genes that normally contain internal 5'-UAG-3' and 5'-UAA-3' codons.