First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA

A 2.4-kb fragment of DNA isolated from the Saccharomyces cerevisiae genome was found to suppress amber mutations when its carrier plasmid was present in high copy number. A 1.2-kb subclone of this fragment was sufficient to confer suppressor activity. Sequencing has established that this fragment carries a normal glutamine tRNA gene. Deletion of this tRNA gene from the subclone resulted in the loss of suppressor activity. The tRNAGln has the anticodon CUG that normally recognizes the glutamine codon CAG. We propose that suppression occurs via an inefficient readthrough of the UAG amber stop codons during translation. Such readthrough requires wobble in the first position of the codon.     

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.     

Site-specific incorporation of non-natural amino acids into proteins in mammalian cells with an expanded genetic code

We describe a detailed protocol for incorporating non-natural amino acids, 3-iodo-L-tyrosine (IY) and p-benzoyl-L-phenylalanine (pBpa), into proteins in response to the amber codon (the UAG stop codon) in mammalian cells. These amino acids, IY and pBpa, are applicable for structure determination and the analysis of a network of protein-protein interactions, respectively. This method involves (i) the mutagenesis of the gene encoding the protein of interest to create an amber codon at the desired site, (ii) the expression in mammalian cells of the bacterial pair of an amber suppressor tRNA and an aminoacyl-tRNA synthetase specific to IY or pBpa and (iii) the supplementation of the growth medium with these amino acids. The amber mutant gene, together with these bacterial tRNA and synthetase genes, is introduced into mammalian cells. Culturing these cells for 16-40 h allows the expression of the full-length product from the mutant gene, which contains the non-natural amino acid at the introduced amber position. This method is implemented using the conventional tools for molecular biology and treating cultured mammalian cells. This protocol takes 5-6 d for plasmid construction and 3-4 d for incorporating the non-natural amino acids into proteins.     

Recessive lethality of yeast strains carrying the SUP61 suppressor results from loss of a transfer RNA with a unique decoding function

A serine-inserting ochre suppressor (SUP61) and its amber allele (SUP-RL1) in the yeast Saccharomyces cerevisiae can only be derived from or maintained in diploid strains heterozygous for the suppressor transfer RNA locus (Brandriss et al., 1975). Two models have been proposed to account for this recessive lethal phenotype. In one, lethality results from the presence of the altered gene product; excessive suppression could interfere with the proper termination of translation. In the second model, lethality is due to the loss of the wild-type function; the suppressor mutation could alter an essential gene that is present in only a single copy in the haploid genome. We have tested a set of specific genetic and biochemical predictions which uniquely distinguish these models.We first isolated several mutant strains carrying second-site mutations which lie within, or are closely linked to, the SUP61 locus. Despite the absence of any biologically detectable suppressor activity, these mutants still give rise to only two viable spores per tetrad. As in the parent, lethality is absolutely correlated with the segregation of the SUP61 allele, and thus it cannot be due solely to suppression.To demonstrate that the SUP61 mutation alters an essential function in haploid cells, a cloned copy of the wild-type gene (sup⁺) was introduced into a diploid containing SUP61 by transformation. Following sporulation, the transformant gave rise to four viable spores per tetrad. We have shown by hybridization analysis that the two spores per tetrad which have suppressor function contain the cloned sup⁺ gene and plasmid DNA integrated in tandem with the SUP61 gene.Piper (1978) has shown that the amber suppressor SUP-RL1 is derived from a tRNA_UCG^Ser gene. More recently, we and others (Etcheverry et al., 1979; Olson et al., 1981; Broach et al., 1981) have provided evidence that the gene coding for this tRNA species exists in only a single copy per haploid genome. Our ability to “cure” the recessive lethal phenotype of SUP61 now allows the conclusion that the gene altered by the suppressor mutation codes for the only isoaccepting species of tRNA^Ser which can decode UCG codons in vivo.     

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.     

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.     

Codon discrimination due to presence of abundant non-cognate competitive tRNA

It has been thought that preferential use of synonymous codons provides high efficiency and fidelity of protein synthesis through specific codon-anticodon interactions. In yeast genes, some codon boxes seem to prefer a codon which is unsuited for its cognate anticodon. Now, we propose that codon usage biases may arise due to presence of abundant non-cognate competitive tRNA capable of misreading a codon by C-U or G-U pairing in the middle position.     

An inducible mammalian amber suppressor: propagation of a poliovirus mutant

We describe a general protocol for controlled gene amplification, which allows conditional expression of high levels of amber suppressor activity in monkey kidney cells, and we demonstrate its use in the genetic analysis of animal viruses by the generation and propagation of the first nonsense mutant of poliovirus. A human amber suppressor tRNASer gene linked to the SV40 origin of replication and a second DNA carrying a temperature-sensitive SV40 large T antigen gene were cotransfected into monkey cells. Cell lines having stably integrated the DNAs were isolated. Shifting the cells from the nonpermissive temperature to a lower permissive temperature caused the amplification of the suppressor tRNA gene, which resulted in suppression efficiencies at amber codons of 50%-70%, as measured by suppression of an amber codon in the E. coli chloramphenicol acetyltransferase gene. A mutant of poliovirus, in which a serine codon in the replicase gene was converted to an amber codon, was efficiently propagated on the suppressor-positive cell lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of approximately 2.5 x 10(-6), surprisingly low for a RNA genome.     

tRNA(2Gln) mutants that translate the CGA arginine codon as glutamine in Escherichia coli.

We present a novel missense suppression system for the selection of tRNA(2GIn) mutants that can efficiently translate the CGA (arginine) codon as glutamine. tRNA(2Gln) mutants were cloned from a partially randomized synthetic gene pool using a plasmid vector that simultaneously expresses the tRNA gene and, to ensure efficient aminoacylation, the glutamine aminoacyl-tRNA synthetase gene (glnS). tRNA mutants that insert glutamine at CGA were selected as missense suppressors of a lacZ mutant (lacZ625(CGA)) that contains CGA substituted for an essential glutamine codon. Preliminary characterizations of four suppressors is presented. All of them contain two anticodon mutations: C-->U at position 34 and U-->C at position 35, which allow for cognate translation of CGA. U35 was previously shown to be an important determinant for glutaminylation of tRNA(2Gln) in vitro; suppression in vivo requires overexpression of the glutaminyl-tRNA synthetase gene (glnS). One tRNA variant contains no further mutations and has the highest missense suppression activity (8%). Three other isolates each contain an additional point mutation that alters suppression efficiency. This system will be useful for further studies of tRNA structure and function. In addition, because relatively efficient translation of the rare CGA codon as glutamine is not toxic for Escherichia coli, it may be possible to translate this sense codon with other alternate meanings, a property which could greatly facilitate protein engineering.     

Development of a yeast system to assay mutational specificity

We have developed a system wherein DNA alterations occurring in a target gene in the yeast Saccharomyces cerevisiae can be determined by DNA sequencing. The target gene, SUP4-o, an ochre suppressor allele of a yeast tyrosine tRNA gene, has been inserted into a shuttle vector (YCpMP2) which is maintained in yeast at a copy number of one per cell Mutations in SUP4-o are selected by virtue of their inactivation of suppressor activity. Rapid DNA preparations from these mutants are used to transform an appropriate bacterial strain. Since YCpMP2 also carries the M13 phage replication origin, superinfection of bacterial cells containing the plasmid with wild-type M13 phage yields single stranded YCpMP2 DNA suitable for dideoxynucleotide chain termination sequencing. We have used this system to examine mutations arising spontaneously in the SUP4-o gene. The spontaneous mutants occurred at a frequency of 3.2 X 10(-6)/viable cell, corresponding to a rate of 2.7 X 10(-7) events/cell division. Following bacterial transformation, 16% of the recovered plasmids tested displayed altered gel mobility consistent with loss of significant portions of the plasmid. Hybridization analysis of total yeast DNA and use of purified YCpMP2 revealed that these very large deletions were not generated in yeast but were associated with bacterial transformation. Among the SUP4-o mutants analyzed by DNA sequencing, we identified each type of single base pair substitution (transitions and transversions), small deletions of varying length (1-32 base pairs) and more extensive deletions of undetermined size. These results demonstrate that the SUP4-o system can be used to detect various types of mutation at numerous sites in a single eukaryotic gene and to characterize the DNA sequence changes responsible for the mutations selected.     

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.     

Vinculin is essential for muscle function in the nematode

Actin filaments in the body wall muscle of the nematode Caenorhabditis elegans are attached to the sarcolemma through vinculin-containing structures called dense bodies, Z-line analogues. To investigate the in vivo function of vinculin, we executed a genetic screen designed to recover mutations in the region of the nematode vinculin gene, deb-1. According to four independent criteria, two of the isolated mutants were shown to be due to alterations in the deb-1 gene. First, antibody staining showed that the mutants had reduced levels of vinculin. Second, the sequence of each mutant gene was altered from that of wild type, with one mutation altering a conserved splice sequence and the other generating a premature amber stop codon. Third, the amber mutant was suppressed by the sup-7 amber suppressor tRNA gene. Finally, injection of a cloned wild type copy of the gene rescued the mutant. Mutant animals lacking vinculin arrested development as L1 larvae. In such animals, embryonic elongation was interrupted at the twofold length, so that the mutants were shorter than wild type animals at the same stage. The mutants were paralyzed and had disorganized muscle, a phenotype consistent with the idea that vinculin is essential for muscle function in the nematode.     

Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs

The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an extrachromosomal array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature dependencies similar to those of endogenous amber suppression. Uaa incorporation efficiency was improved by impairing the nonsense-mediated mRNA decay pathway through knockdown of smg-1. We have generated stable transgenic worms capable of genetically encoding Uaas, enabling Uaa exploitation to address complex biological problems within a metazoan. We anticipate our strategies will be generally extendable to other multicellular organisms.     

Principles of start codon recognition in eukaryotic translation initiation

Selection of the correct start codon during initiation of translation on the ribosome is a key event in protein synthesis. In eukaryotic initiation, several factors have to function in concert to ensure that the initiator tRNA finds the cognate AUG start codon during mRNA scanning. The two initiation factors eIF1 and eIF1A are known to provide important functions for the initiation process and codon selection. Here, we have used molecular dynamics free energy calculations to evaluate the energetics of initiator tRNA binding to different near-cognate codons on the yeast 40S ribosomal subunit, in the presence and absence of these two initiation factors. The results show that eIF1 and eIF1A together cause a relatively uniform and high discrimination against near-cognate codons. This works such that eIF1 boosts the discrimination against a first position near-cognate G-U mismatch, and also against a second position A-A base pair, while eIF1A mainly acts on third codon position. The computer simulations further reveal the structural basis of the increased discriminatory effect caused by binding of eIF1 and eIF1A to the 40S ribosomal subunit.     

Non-standard translational events in Candida albicans mediated by an unusual seryl-tRNA with a 5'-CAG-3' (leucine) anticodon.

From in vitro translation studies we have previously demonstrated the existence of an apparent efficient UAG (amber) suppressor tRNA in the dimorphic fungus Candida albicans (Santos et al., 1990). Using an in vitro assay for termination codon readthrough the tRNA responsible was purified to homogeneity from C.albicans cells. The determined sequence of the purified tRNA predicts a 5'-CAG-3' anticodon that should decode the leucine codon CUG and not the UAG termination codon as originally hypothesized. However, the tRNA(CAG) sequence shows greater nucleotide homology with seryl-tRNAs from the closely related yeast Saccharomyces cerevisiae than with leucyl-tRNAs from the same species. In vitro tRNA-charging studies demonstrated that the purified tRNA(CAG) is charged with Ser. The gene encoding the tRNA was cloned from C.albicans by a PCR-based strategy and DNA sequence analysis confirmed both the structure of the tRNA(CAG) and the absence of any introns in the tRNA gene. The copy number of the tRNA(CAG) gene (1-2 genes per haploid genome) is in agreement with the relatively low abundance (< 0.5% total tRNA) of this tRNA. In vitro translation studies revealed that the purified tRNA(CAG) could induce apparent translational bypass of all three termination codons. However, peptide mapping of in vitro translation products demonstrated that the tRNA(CAG) induces translational misreading in the amino-terminal region of two RNA templates employed, namely the rabbit alpha- and beta-globin mRNAs. These results suggest that the C.albicans tRNA(CAG) is not an 'omnipotent' suppressor tRNA but rather may mediate a novel non-standard translational event in vitro during the translation of the CUG codon. The possible nature of this non-standard translation event is discussed in the context of both the unusual structural features of the tRNA(CAG) and its in vitro behaviour.