Inhibition of Growth by Amber Suppressors in Yeast

Strains of the yeast Saccharomyces cerevisiae that contain highly efficient amber (UAG) suppressors grow poorly on nutrient medium, while normal or nearly normal growth rates are observed when these strains lose the supressors or when the suppressors are mutated to lower efficiencies. The different growth rates account for the accumulation of mutants with lowered efficiencies in cultures of strains with highly efficient amber suppressors. Genetic analyses indicate that one of the mutations with a lowered efficiency of suppression is caused by an intragenic mutation of the amber supressor. The inhibition of growth caused by excessive suppression is expected to be exacerbated when appropriate suppressors are combined together in haploid cells if two suppressors act with a greater efficiency than a single suppressor. Such retardation of growth is observed with combinations of two UAA (ochre) suppressors (Gilmore 1967) and with combinations of two UAG suppressors when the efficiencies of each of the suppressors are within a critical range. In contrast, combinations of a UAA suppressor and a UAG suppressor do not affect growth rate. Apparently while either excessive UAA or excessive UAG suppression is deleterious to yeast, a moderate level of simultaneous UAA and UAG suppression is not.     

Isolation and Characterization of Amber Suppressors in Yeast

Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome c in cyc1-179 strains suppressed with these efficient amber suppressors.     

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     

Studies on the bacteriophage MS2: XLI. Nature of the azure mutation

Azure (or reverse amber) mutants grow normally on wild-type Escherichia coli but not on host strains harbouring a strong UAG suppressor mutation. Three different bacteriophage MS2 azure mutants obtained by treatment with nitrous acid have been characterized at the nucleotide sequence level. The 3′-end fragment of the ³²P-labelled mutant genomes was isolated by DNA:RNA hybridization and treatment with nuclease S₁, and was analyzed by mini-fingerprinting of the RNA. It is known that the wild-type MS2 polymerase gene ends with a UAG codon, followed seven triplets further by an in-phase UAA triplet. All three azure mutants contained an A → G transition in this UAA second stop codon of the polymerase gene, resulting in a second suppressible UAG (amber) codon. Analysis of revertants demonstrated that the azure mutation can be counteracted either by a true site reversion at the second stop or by the creation of a new stop signal for the polymerase gene, either UAA (ochre) or UGA (opal), before or at the first stop, or beyond the second stop. On the basis of these results, a mechanism for the azure mutation is proposed. Silent mutations (one in the coding region, three in the untranslated 3′-terminal sequence) have also been observed in these phage stocks.     

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.     

Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli

Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing. In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon. The resultant suppressor tRNA, Su79, is a very strong amber suppressor. Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing. These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon. The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor. The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification. The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.     

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

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

Determination of mutation rates in bacteriophage T4 by unneighborly base pairs: Genetic analysis

Earlier studies showed that the 2-aminopurine-induced mutation rate at a particular base pair can be influenced by the base adjacent to, or one additional base-pair removed from, the measured site (Koch, 1971). The present study extends to 0.3 map unit (about 30 base pairs) the distance at which a single base-pair substitution can exert such an effect. A particular base-pair substitution (defined as a ts mutation in the rIIA gene of bacteriophage T4) reduces the spontaneous, 2-aminopurine-induced and nitrous acid-induced reversions of an rIIA amber mutation approximately threefold. The ts mutation also reduces the 2-aminopurine-induced conversion of the corresponding ochre codon to amber (UAA → UAG) about twofold and to opal (UAA → UGA) about eightfold. The 2-aminopurine-induced reversion of the ochre codon to a glutamine codon (UAA → CAA), however, is not affected. Control experiments demonstrate that these observed reductions in mutation frequency do not result from unacceptable pathways of reversion in the presence of the ts allele.     

Context effects: translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message

The efficiency of various suppressor tRNAs in reading the UAG amber codon has been measured at 42 sites in the lacI gene. Results indicate that: (1) for all suppressors, efficiency is not an a priori value; rather, it is determined at each site by the specific reading context of the suppressed codon; (2) the degree of sensitivity to context effects differs among suppressors. Most affected is amber suppressor supE (su2), whose activity varies over a 20-fold range depending on context; (3) context effects are produced by residues present at the 3' side of the UAG codon. The most important role appears to be played by the base that is immediately adjacent to the codon. When this base is a purine, the amber codon is suppressed more efficiently than when a pyrimidine is in the same position. Superimposed on this initial pattern, the influence of bases further downstream to the UAG triplet can be detected also. The possibility is discussed that context effects are produced by the whole codon following UAG in the message.     

Isolation and characterization of context mutations affecting the suppressibility of nonsense mutations

Summary Secondary mutations which increase the efficiency of suppression of nonsense mutations in the rHB cistron of bacteriophage T4 have been isolated. These secondary mutations, called context mutations, map at sites very close to the nonsense codon, possibly on the promotor distal side. In context-nonsense double mutants, the amount of suppressed gene product is increased approximately 10-fold. The context mutations examined can act on the UAA (ochre) nonsense allele as well as on the UAG (amber) nonsense allele at a given site. These context mutations affect all suppression mechanisms analyzed (genetic suppressors. 5-fluorouracil suppression and spontaneous suppression).We suggest that context mutations affect information which is significant to the termination of polypeptide chains. According to our view, context mutations change the immediate neighborhood of nonsense mutations and so reduce the degree of resemblance to the sequences normally used for the termination of translation.     

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.     

Variation of Mutagenic Action on Nonsense Mutants at Different Sites in the Iso-1-Cytochrome c Gene of Yeast

Three ochre and two amber mutants in yeast have been definitively identified by the amino acid replacements in iso-1-cytochromes c from intragenic revertants. Except for rare and sometimes unusual changes, all of the replacements were single amino acids whose codons differed from UAA or UAG by one base. These assignments, which were based on the absence of tryptophan replacements in ochre revertants, could be corroborated from the studies of two groups of suppressors that were shown to act on either the ochre or amber mutants. All five nonsense mutants are located at different sites in the cyc1 gene and all are at sites that can be occupied by amino acids having a wide range of structures. The relative frequencies of the amino acid replacements indicate that identical codons located at different sites may respond differently to a mutagenic agent. Notably glutamine replacements occurred almost exclusively in UV-induced revertants of only one ochre mutant cyc1–9, but not at all or at reduced proportions in the others. Similarly, lysine replacements occurred almost exclusively in the NA-induced revertants of only the ochre mutant cyc1–72, but not at all in the others. These and other results reveal that mutation of A·T base pairs by UV and nitrous acid are dependent upon the location of the codon within the gene as well as the location of the base pair within the codon. From these findings, it appears as if the type of base-pair changes induced by UV and nitrous acid are strongly influenced by adjacent nucleotide sequences.     

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.     

UV-mutagenesis at a cloned target sequence: converted suppressor mutation is insensitive to mutation frequency decline regardless of the gene orientation

Premutational lesions produced by ultraviolet radiation in the Gln2 tRNA genes of E. coli B/r show differing sensitivities to a mutation avoidance phenomenon known as mutation frequency decline (MFD). A mutation event that changes the wild-type gene to an amber (UAG) suppressor is normally sensitive to MFD. Mutation of this amber suppressor to an ochre (UAA) suppressor is not sensitive to MFD. These two mutation events occur in the same anticodon region of the DNA. The dissimilarity of MFD sensitivity between these two mutations may result because the respective premutational photoproducts for the two are located in opposite strands of duplex DNA. To examine the effect of strand position of the premutational lesions on MFD, recombinant lambda phage were constructed that contained the amber suppressor as a mutation target in the two possible orientations. Comparison of MFD in bacterial lysogens containing either of the two types of recombinant prophage indicated that reversing the orientation of the target sequence relative to adjacent bacterial DNA had no effect on MFD. Since rotational inversion of the target sequence did not alter the sensitivity to MFD of mutation occurring at the cloned target gene, the antimutation process inherent to MFD can not be attributed to an asymmetrical interaction between the template strands and the DNA-replication complex.     

Tyrosine substitutions resulting from suppression of amber mutants of iso-1-cytochrome c in yeast

The suppressors SUP6-2 and SUP7-2 can cause the production of approxi- mately 25 to 60% of the normal amount of iso-1-cytochrome c when they are coupled to the amber (UAG) mutants cy1–179 and cy1–76. The iso-1-cytochromes c contain residues of tyrosine at the positions which correspond to the sites of the amber codons. SUP6-2 and SUP7-2 do not suppress ochre (UAA) mutants. The SUP6-2 and the SUP7-2 genes are apparently alleles of the SUP6-1 and SUP7-1 genes, respectively, which cause the insertion of tyrosine at ochre (UAA) codons (ochre-specific suppressors). It is suggested that the gene products of the allelic amber suppressors and ochre-specific suppressors (the SUP6-1 and SUP6-2 suppressors and theSUP7-1 andSUP7-2 suppressors) are two differently altered forms of the same tyrosine tRNA.     

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.     

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.     

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.     

Efficient decoding of the UAG triplet as a full-fledged sense codon enhances the growth of a prfA-deficient strain of Escherichia coli

We previously reassigned the amber UAG stop triplet as a sense codon in Escherichia coli by expressing a UAG-decoding tRNA and knocking out the prfA gene, encoding release factor 1. UAG triplets were left at the ends of about 300 genes in the genome. In the present study, we showed that the detrimental effect of UAG reassignment could be alleviated by increasing the efficiency of UAG translation instead of reducing the number of UAGs in the genome. We isolated an amber suppressor tRNA(Gln) variant displaying enhanced suppression activity, and we introduced it into the prfA knockout strain, RFzero-q, in place of the original suppressor tRNA(Gln). The resulting strain, RFzero-q3, translated UAG to glutamine almost as efficiently as the glutamine codons, and it proliferated faster than the parent RFzero-q strain. We identified two major factors in this growth enhancement. First, the sucB gene, which is involved in energy regeneration and has two successive UAG triplets at the end, was expressed at a higher level in RFzero-q3 than RFzero-q. Second, the ribosome stalling that occurred at UAG in RFzero-q was resolved in RFzero-q3. The results revealed the importance of "backup" stop triplets, UAA or UGA downstream of UAG, to avoid the deleterious impact of UAG reassignment on the proteome.