Release factor competition is equivalent at strong and weakly suppressed nonsense codons

Summary We have compared the competition between strong or weak suppressor tRNAs and translational release factors (RF) at nonsense codons in the lacI gene of Escherichia coli. Using the F'lacIZ fusions developed by Miller and coworkers, UAG, UAA, and UGA codons at positions 189 and 220 were efficiently suppressed by plasmid-borne tRNAtrp suppressors cognate to each nonsense triplet. Introduction of a compatible RF 1 plasmid competed at UAG and UAA but not UGA codons. An RF2 expressing plasmid competed at UAA and UGA but had little effect at UAG. Release factor competition against weak suppressors was measured using combinations of noncognate suppressors and nonsense codons. In each case, release factor plasmids behaved identically towards poorly suppressed codons as they did when the same codons were efficiently suppressed. The implications for these studies on the role of release factors in nonsense suppression context effects are discussed.     

Mutations to nonsense codons in human genetic disease: implications for gene therapy by nonsense suppressor tRNAs.

Nonsense suppressor tRNAs have been suggested as potential agents for human somatic gene therapy. Recent work from this laboratory has described significant effects of 3' codon context on the efficiency of human nonsense suppressors. A rapid increase in the number of reports of human diseases caused by nonsense codons, prompted us to determine how the spectrum of mutation to either UAG, UAA or UGA codons and their respective 3' contexts, might effect the efficiency of human suppressor tRNAs employed for purposes of gene therapy. This paper presents a survey of 179 events of mutations to nonsense codons which cause human germline or somatic disease. The analysis revealed a ratio of approximately 1:2:3 for mutation to UAA, UAG and UGA respectively. This pattern is similar, but not identical, to that of naturally occurring stop codons. The 3' contexts of new mutations to stop were also analysed. Once again, the pattern was similar to the contexts surrounding natural termination signals. These results imply there will be little difference in the sensitivity of nonsense mutations and natural stop codons to suppression by nonsense suppressor tRNAs. Analysis of the codons altered by nonsense mutations suggests that efforts to design human UAG suppressor tRNAs charged with Trp, Gln, and Glu; UAA suppressors charged with Gln and Glu, and UGA suppressors which insert Arg, would be an essential step in the development of suppressor tRNAs as agents of human somatic gene therapy.     

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).     

Neurospora crassa supersuppressor mutants are amber codon-specific

Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.     

A novel method for detection and characterization of ochere suppressors in Escherichia coli

Summary We have found a new method for specifically detecting the occurrence of ochre (UAA) suppression in Escherichia coli. It is based on a procedure we used several years ago to distinguish trpA missense mutants from nonsense mutants, and relies on the generally low efficiency of suppression that seems to be characteristic of ochre suppressors in E. coli. Suppressed ochre mutants are distinguishable from trpA revertants by their inability to grow on glucose minimal medium containing a low concentration (1.5 m/ml) of indole and a high concentration (50 g/ml) of 5-methyl-DL-tryptophan (Ind-5MT). The procedure provides a specific and rapid means for detection of UAA derived from missense codons and has also been exploited to obtain different classes of ochre suppressors derived from the amber suppressor supDam and from a glycine tRNA missense suppressor. The Ind-5MT phenotype seems to depend in some way on the location of the ochre codon within the trpA messenger RNA. The method can be put to many uses and should be generally applicable to all low-efficiency nonsense suppressors, including those specific for UAG and UGA.Preliminary reports of portions of this work were presented at the spring meeting of the Texas Branch of the American Society for Microbiology, College Station, Texas, March, 1975     

Patterns of Genetic and Phenotypic Suppression of lys2 Mutations in the Yeast SACCHAROMYCES CEREVISIAE

A total of 358 lys2 mutants of Saccharomyces cerevisiae have been characterized for suppressibility by the following suppressors: UAA and UAG suppressors that insert tyrosine, serine or leucine; a putative UGA suppressor; an omnipotent suppressor SUP46; and a frameshift suppressor SUF1–1. In addition, the lys2 mutants were examined for phenotypic suppression by the aminoglycoside antibiotic paromomycin, for osmotic remediability and for temperature sensitivity. The mutants exhibited over 50 different patterns of suppression and most of the nonsense mutants appeared similar to nonsense mutants previously described. A total of 24% were suppressible by one or more of the UAA suppressors, 4% were suppressible by one or more of the UAG suppressors, while only one was suppressible by the UGA suppressor and only one was weakly suppressible by the frameshift suppressor. One mutant responded to both UAA and UAG suppressors, indicating that UAA or UAG mutations at certain rare sites can be exceptions to the specific action of UAA and UAG suppressors. Some of the mutants appeared to require certain types of amino acid replacements at the mutant sites in order to produce a functional gene product, while others appeared to require suppressors that were expressed at high levels. Many of the mutants suppressible by SUP46 and paromomycin were not suppressible by any of the UAA, UAG or UGA suppressors, indicating that omnipotent suppression and phenotypic suppression need not be restricted to nonsense mutations. All of the mutants suppressible by SUP46 were also suppressible by paromomycin, suggesting a common mode of action of omnipotent suppression and phenotypic misreading.     

Analysis of specific misreading in Escherichia coli

The pattern of specific misreading by nonsense suppressors has been investigated using nonsense mutants in the rIIB gene of phage T4 and in the lacZ gene of Escherichia coli. It is shown that a su⁺ transfer RNA which reads UAG also misreads UAA but not UGA, a su⁺ tRNA which reads UAA (while it also reads UAG by wobble) misreads UGA and a su⁺ tRNA which reads UGA also probably misreads UAA but not UAG.These specific types of errors in translation occur in the absence of streptomycin. The addition of the drug raises their level without altering the pattern described. A ribosomal mutation str A reduces the level of specific misreading; by contrast, a ram mutation strongly increases this level. In all cases the specific pattern is not affected.The rate of specific misreading of nonsense codons in different cases ranges from less than 0.001% to more than 3%. Since the frequency of misreading is sitespecific (unpublished observations), the rates obtained cannot be extrapolated to any other codon at any other site.     

Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.Translation termination is mediated by one of the three stop codons (UAA, UAG, or UGA). When such stop codons arise in coding sequences due to mutations, referred to as nonsense mutations, they lead to abrupt arrest of the translation process. However, the termination efficiency of such nonsense codons is not 100%, as certain tRNAs have the ability to read these nonsense codons. Genetic code ambiguity is seen in several organisms. Stop codons have been shown to have alternate roles apart from translation termination. In organisms from all three domains of life, UGA encodes selenocysteine through a specialized mechanism. In Methanosarcinaceae, UAG encodes pyrrolysine (3). UAA and UAG are read as glutamine codons in some green algae and ciliates such as Tetrahymena and Diplomonads (24), and UAG alone encodes glutamine in Moloney murine leukemia virus (32). UGA encodes cysteine in Euplotes; tryptophan in some ciliates, Mycoplasma species, Spiroplasma citri, Bacillus, and tobacco rattle virus; and an unidentified amino acid in Pseudomicrothorax dubius and Nyctotherus ovalis (30). In certain cases the context of the stop codon in translational readthrough has been shown to play a role; for example, it has been reported that in vitro in tobacco mosaic virus, UAG and UAA are misread by tRNA^Tyr in a highly context-dependent manner (34, 9).Termination suppressors are of three types, i.e., amber, ochre, and opal suppressors, which are named based on their ability to suppress the three stop codons. Amber suppressors can suppress only amber codons, whereas ochre suppressors can suppress ochre codons (by normal base pairing) as well as amber codons (by wobbling) and opal suppressors can read opal and UGG tryptophan codon in certain cases. As described by Sambrook et al. (27), a few amber suppressors can also suppress ochre mutations by wobbling. The suppression efficiency varies among these suppressors, with amber suppressors generally showing increased efficiency over ochre and opal suppressors. supE44, an amber suppressor tRNA, is an allele of and is found in many commonly used strains of Escherichia coli K-12. Earlier studies have shown that supE44 is a weak amber suppressor and that its efficiency varies up to 35-fold depending on the reading context of the stop codon (8).Translational accuracy depends on several factors, which include charging of tRNAs with specific amino acids, mRNA decoding, and the presence of antibiotics such as streptomycin and mutations in ribosomal proteins which modulate the fidelity of the translational machinery. Among these, mRNA decoding errors have been reported to occur at a frequency ranging from about 10⁻³ to 10⁻⁴ per codon. Translational misreading errors also largely depend on the competition between cognate and near-cognate tRNA species. Poor availability of cognate tRNAs increases misreading (18).Several studies with E. coli and Saccharomyces cerevisiae have shown the readthrough of nonsense codons in suppressor-free cells. In a suppressor-free E. coli strain, it has been shown in vitro that glutamine is incorporated at the nonsense codons UAG and UAA (26). It has been reported that overexpression of wild-type tRNA^Gln in yeast suppresses amber as well as ochre mutations (25). In this study, we have confirmed the presence of an amber suppressor mutation in the glnX gene in a supE44 strain by sequence analysis. This was done essentially because we observed that supE44 could also suppress lacZ ochre mutations, albeit inefficiently. On further investigation using an in vivo luciferase reporter assay system for tRNA-mediated nonsense suppression (28), we found that the efficiency of suppression of amber lesion by supE44 is significantly higher than that reported previously in the literature. An increased ability to suppress ochre and opal nonsense mutations was observed in cells bearing supE44 compared to in the wild type. Such an effect was observed only in the stationary phase and was abolished in logarithmic phase.     

Suppression of UAA and UGA termination codons in mutant murine leukemia viruses.

Genomes of mammalian type C retroviruses contain a UAG termination codon between the gag and pol coding regions. The pol region is expressed in the form of a gag-pol fusion protein following readthrough suppression of the UAG codon. We have used oligonucleotide-directed mutagenesis to change the UAG in Moloney murine leukemia virus to UAA or UGA. These alternate termination codons were also suppressed, both in infected cells and in reticulocyte lysates. Thus, the signal or context inducing suppression of UAG in wild-type Moloney murine leukemia virus is also effective with UAA and UGA. Further, mammalian cells and cell extracts contain tRNAs capable of translating UAA and UGA as amino acids. To our knowledge, this is the first example of natural suppression of UAA in higher eucaryotes.     

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors

Summary A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.The mutated gene (uar) has been mapped and found to be recessive both with respect to suppressor-enhancing ability as well as for temperature sensitivity. The phenotype is partly suppressed by the ochre suppressor SuC. It is suggested that uar codes for a protein, which is involved in translational termination at UAG and UAA stop codons.     

The influence of the reading context upon the suppression of nonsense codons

Summary We have examined the response of phage T4 nonsense mutations located at various sites within the same cistron to different suppression agents. A wide range of suppression efficiency is found for both ochre (UAA) and amber (UAG) mutations under conditions where suppression provides a measurement of the amount of chain propagation past the mutated site. We have established a relationship between our measurement-the size of the phage yield-and the amount of rIIB product present in the infection. Our data suggest that the 1000-fold range of variations in yields observed in the rIIB cistron corresponds to a 30-fold range of variation in the level of rIIB product, i.e. in the relative frequency of chain propagation past the various nonsense codons included in our test.From the parallelism of response of any particular mutant to very different suppression mechanisms we conclude that the efficiency of suppression is site specific, that is to say, that the main factor determining the frequency of chain propagation at a nonsense codon by any type of suppression mechanism is the nucleotide sequence adjacent to the nonsense codon (reading context).We propose that the recognition of a natural termination signal involves a sequence longer than a nonsense codon and that nonsense codons outside of their natural environment induce variable termination rates which are reflected in the suppression potential.     

Variation of phenotypic suppression due to the psi+ and psi- extrachromosomal determinants in yeast.

Paromomycin, an aminoglycoside antibiotic, can phenotypically suppress nonsense mutations in the yeast Saccharomyces cerevisiae (Palmer et al., 1979). We report here that the extrachromosomal determinant, ψ⁺, enhances this phenotypic suppression of all three nonsense mutations. UAG. UAA and UGA, by three-to sevenfold.     

Mutations in E.coli 16s rRNA that enhance and decrease the activity of a suppressor tRNA.

The in vivo expression of mutations constructed within helix 34 of 16S rRNA has been examined together with a nonsense tRNA suppressor for their action at stop codons. The data revealed two novel results: in contrast to previous findings, some of the rRNA mutations affected suppression at UAA and UAG nonsense codons. Secondly, both an increase and a decrease in the efficiency of the suppressor tRNA were induced by the mutations. This is the first report that rRNA mutations decreased the efficiency of a suppressor tRNA. The data are interpreted as there being competition between the two release factors (RF-1 and RF-2) for an overlapping domain and that helix 34 influences this interaction.     

Construction, expression, and function of a new yeast amber suppressor, tRNATrpA

The number of different tRNA species in Saccharomyces cerevisiae known to be capable of suppressing termination of translation at UAG, UAA, and UGA codons is limited to those which insert tyrosine, leucine, and serine. Suppressor tRNAs that insert other amino acids, even those whose anticodons differ from the expected recognition sequences for nonsense codons by a single nucleotide, have never been identified via classical genetic analysis. We have used site-directed mutagenesis to convert the anticodon of a cloned tRNATrp gene from CCA to CTA with the expectation that this gene would produce tRNA molecules capable of interacting with the UAG terminator codon. We show that this form of the gene can be transcribed and spliced in vitro to produce mature tRNA with the expected base sequence. The putative suppressor gene has been introduced into several S. cerevisiae host strains using the centromere vector YCp19. Efficient suppression of amber mutations met8-1, tyr7-1, and lys2-801 results from the presence of the CTA form of tDNATrp. Two UAA mutants, leu2-1 and ade2-101, and the UGA marker his4-260 are not suppressed.     

Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.     

Some Aspects of E. coli Promoter Evolution Observed in a Molecular Evolution Experiment

We devised a molecular evolution procedure to evolve E. coli promoter sequences and applied it to observe an arbitrary, nonfunctional sequence evolving into functional promoters. In the experiments, DNA sequence variations were generated with error-prone PCR and were inserted in the promoter region of the cat (chloramphenicol acetyl transferase) gene on a plasmid. Upon transforming the cells, functional promoters on the plasmid were selected according to the chloramphenicol resistance. Within a few cycles of mutation-selection, promoters emerged, and the sequences converged into a small number of groups. In the process, the extended minus 10 type of promoters emerged quickly, and small deletions were often involved in adjusting the length between the −35 and the −10 elements. Our results also suggest a possible selection for promoter stability against mutation. [Reviewing Editior: Dr. Laura Landweber]     

Construction and testing of a bacterial luciferase reporter gene system forin Vivo measurement of nonsense suppression inStreptomyces

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.     

Suppression of Nonsense Mutations Induced by Expression of an RNA Complementary to a Conserved Segment of 23S rRNA

We identified a short RNA fragment, complementary to the Escherichia coli 23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.     

The influence of the reading context upon the suppression of nonsense codons

Summary One of the basic assumptions of the current views of the genetic code is that the translation machinery reads the messenger RNA one nucleotide triplet codon at a time and that the meaning of a particular codon should not be effected by the surrounding nucleotide sequence (the reading context). Reexamination of existing data shows that this assumption does not hold for the case of suppression of the nonsense codons UAG (amber) and UAA (ochre).The efficiency of amino acid insertion in response to these nonsense codons appears to strongly depend on their location within the message. It is suggested that the translation machinery may interact with a nucleotide sequence longer than three nucleotides when involved in a chain termination reaction.