Nonsense Mutants in the rII A Cistron of Bacteriophage T4

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.     

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 Since the frequency of misreading is sitespecific (unpublished observations), the rates obtained cannot be extrapolated to any other codon at any other site.     

Differentiation between Amber and Ochre Mutants of Yeast by Reversion with 4-Nitroquinoline-1-Oxide

4-nitroquinoline-1-oxide (NQO) induces high frequencies of intragenic revertants of amber (UAG) but not ochre (UAA) mutants of yeast. Distinction of the amber and ochre codons was made with well-characterized nonsense mutants of the iso-1-cytochrome c gene (cyc1 mutants) as well as with nonsense mutants having nutritional requirements. Thus the NQO-induced reversion frequencies corroborated the assignments that were based on the pattern of amino acid replacements in intragenic revertants and on the speficity of suppression. It was concluded from these results and from the results of a previous investigation with other cyc1 mutants (Prakash, Stewart and Sherman 1974) that NQO induces transversions of G:C base pairs at many sites and that the specificity is not strongly influenced by neighboring base pairs in at least the strains examined in these studies. NQO was previously shown to induce G:C → A:T transitions at least at one site and this and the previous study established that it does not significantly mutate A:T base pairs at numerous sites. Thus NQO can be used to selectively mutate G:C base pairs and to determine if the pathways of reverse mutations involve G:C base pairs. Suppressors that act on either amber or ochre mutants were induced with NQO, indicating that they can arise by mutations of G:C base pairs.     

Recombination in phage T4 gene-43 (DNA polymerase) mutants.

Summary The effect of phage T4 gene 43 (DNA polymerase) mutations on recombination between adjacent base pairs was measured in rII amber and opal mutants.The mutator allele tsL56 did not promote recombination frequencies at the two sites in which its effect was studied. The antimutator allele tsCB87 caused slight or no reduction in recombination frequencies at five sites.Abbreviations A, T, G and C are adenine, thymine, guanine and 5-hydroxymethylcytosine, respectively     

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.     

Marker Effects on Reversion of T4rII Mutants

The frequencies of 2-aminopurine- and 5-bromouracil-induced A:T leads to G:C transitions were compared at nonsense sites throughout the rII region of bacteriophage T4. These frequencies are influenced both by adjacent base pairs within the nonsense codons and by extracodonic factors. Following 2AP treatment, they are high in amber (UAG) and lower in opal (UGA) codons than in allelic ochre (UAA) codons. In general, 5BU-induced transitions are more frequent in both amber and opal codons than in the allelic ochre codons. 2AP- and 5BU-induced transition frequencies in the first and third positions of opal codons are correlated with those in the corresponding positions of the allelic ochre codons. Similarly, the frequencies of 2AP-induced transition in the first and second positions of amber codons and their ochre alleles are correlated. However, there is little correlation between the frequencies of 5BU-induced transitions in the first and second positions of allelic amber and ochre codons.     

Connection between stop codon reassignment and frequent use of shifty stop frameshifting

Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recently demonstrated that Euplotes octocarinatus peptide release factor eRF1 ignores UGA termination codons while continuing to recognize UAA and UAG. Here we show that both the Tetrahymena thermophila and E. octocarinatus eRF1 factors allow efficient frameshifting at all three termination codons, suggesting that UGA redefinition also impaired UAA/UAG recognition. Mutations of the Euplotes factor restoring a phylogenetically conserved motif in eRF1 (TASNIKS) reduced programmed frameshifting at all three termination codons. Mutation of another conserved residue, Cys124, strongly reduces frameshifting at UGA while actually increasing frameshifting at UAA/UAG. We will discuss these results in light of recent biochemical characterization of these mutations.     

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.     

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.     

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.     

Consequences of frameshift mutations in the trp A, trp B and lac I genes of Escherichia coli and in Salmonella typhimurium

There are two types of frameshift mutation so far as out-of-phase reading of the genetic message is concerned. Firstly there are those frameshifts which result from the loss of one, or the addition of two adjacent, base pairs (?1(+2) type). Secondly there are those which result from the loss of two, or the addition of 1, base pair, in the DNA (?2(+1) type). We have compared the qualitative and quantitative consequences of these two types of frameshift upon the gene products of the lac I gene of E. coli and the trp A and trp B genes of E. coli and S. typhimurium. Both types of frameshift produce predominantly opal (UGA) rather than UAA (ochre) or UAG (amber) premature stop codons. Surprisingly the ?2(+1) type of frameshift leads to very few AUG or GUG reinitiation codons compared with the other, ?1(+2), type of frameshift.     

Serine insertion caused by the ribosomal suppressor SUP46 in yeast

The ribosomal suppressor SUP46 isolated from the yeast Saccharomyces cerevisiae suppresses a broad range of mutations, including at least some UAA, UAG and UGA alleles. The SUP46 suppressor causes the insertion of serine into iso-1-cytochrome c at the site of the UAA mutation in the cyc1-72 allele. It is believed that the altered ribosomes in the SUP46 suppressor allow a serine tRNA to misread UAA codons.     

Origin of an Alternative Genetic Code in the Extremely Small and GC–Rich Genome of a Bacterial Symbiont

The genetic code relates nucleotide sequence to amino acid sequence and is shared across all organisms, with the rare exceptions of lineages in which one or a few codons have acquired novel assignments. Recoding of UGA from stop to tryptophan has evolved independently in certain reduced bacterial genomes, including those of the mycoplasmas and some mitochondria. Small genomes typically exhibit low guanine plus cytosine (GC) content, and this bias in base composition has been proposed to drive UGA Stop to Tryptophan (Stop→Trp) recoding. Using a combination of genome sequencing and high-throughput proteomics, we show that an α-Proteobacterial symbiont of cicadas has the unprecedented combination of an extremely small genome (144 kb), a GC–biased base composition (58.4%), and a coding reassignment of UGA Stop→Trp. Although it is not clear why this tiny genome lacks the low GC content typical of other small bacterial genomes, these observations support a role of genome reduction rather than base composition as a driver of codon reassignment.     

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.     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

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.     

Origin of an Alternative Genetic Code in the Extremely Small and GC–Rich Genome of a Bacterial Symbiont

The genetic code relates nucleotide sequence to amino acid sequence and is shared across all organisms, with the rare exceptions of lineages in which one or a few codons have acquired novel assignments. Recoding of UGA from stop to tryptophan has evolved independently in certain reduced bacterial genomes, including those of the mycoplasmas and some mitochondria. Small genomes typically exhibit low guanine plus cytosine (GC) content, and this bias in base composition has been proposed to drive UGA Stop to Tryptophan (Stop→Trp) recoding. Using a combination of genome sequencing and high-throughput proteomics, we show that an α-Proteobacterial symbiont of cicadas has the unprecedented combination of an extremely small genome (144 kb), a GC–biased base composition (58.4%), and a coding reassignment of UGA Stop→Trp. Although it is not clear why this tiny genome lacks the low GC content typical of other small bacterial genomes, these observations support a role of genome reduction rather than base composition as a driver of codon reassignment.     

Defined set of cloned termination suppressors: in vivo activity of isogenetic UAG, UAA, and UGA suppressor tRNAs.

We have cloned an isogenetic set of UAG, UAA, and UGA suppressors. These include the Su7 -UAG, Su7 -UAA, and Su7 -UGA suppressors derived from base substitutions in the anticodon of Escherichia coli tRNATrp and also Su9 , a UGA suppressor derived from a base substitution in the D-arm of the same tRNA. These genes are cloned on high-copy-number plasmids under lac promoter control. The construction of the Su7 -UAG plasmid and the wild-type trpT plasmid have been previously described ( Yarus , et al., Proc. Natl. Acad. Sci. U.S.A. 77:5092-5097, 1980). Su7 -UAA ( trpT177 ) is a weak suppressor which recognizes both UAA and UAG nonsense codons and probably inserts glutamine. Su7 -UGA ( trpT176 ) is a strong UGA suppressor which may insert tryptophan. Su9 ( trpT178 ) is a moderately strong UGA suppressor which also recognizes UGG (Trp) codons, and it inserts tryptophan. The construction of these plasmids is detailed within. Data on the DNA sequences of these trpT alleles and on amino acid specificity of the suppressors are presented. The efficiency of the cloned suppressors at certain nonsense mutations has been measured and is discussed with respect to the context of these codons.     

Mediation of the A/B-DNA helix transition by G-tracts in the crystal structure of duplex CATGGGCCCATG

The crystal structure of the DNA dodecamer duplex CATGGGCCCATG lies on a structural continuum along the transition between A- and B-DNA. The dodecamer possesses the normal vector plot and inclination values typical of B-DNA, but has the crystal packing, helical twist, groove width, sugar pucker, slide and x-displacement values typical of A-DNA. The structure shows highly ordered water structures, such as a double spine of water molecules against each side of the major groove, stabilizing the GC base pairs in an A-like conformation. The different hydration of GC and AT base pairs provides a physical basis for solvent-dependent facilitation of the A↔B helix transition by GC base pairs. Crystal structures of CATGGGCCCATG and other A/B-DNA intermediates support a ‘slide first, roll later’ mechanism for the B→A helix transition. In the distribution of helical parameters in protein–DNA crystal structures, GpG base steps show A-like properties, reflecting their innate predisposition for the A conformation.     

Rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons

A stop or nonsense codon is an in-frame triplet within a messenger RNA that signals the termination of translation. One common feature shared among all three nonsense codons (UAA, UAG, and UGA) is a uridine present at the first codon position. It has been recently shown that the conversion of this uridine into pseudouridine (Ψ) suppresses translation termination, both in vitro and in vivo. Furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two specific amino acids in a nonsense codon-dependent fashion. Ψ differs from uridine by a single N1H group at the C5 position; how Ψ suppresses termination and, more importantly, enables selective decoding is poorly understood. Here, we provide molecular rationales for how pseudouridylated stop codons are selectively decoded. Our analysis applies crystal structures of ribosomes in varying states of translation to consider weakened interaction of Ψ with release factor; thermodynamic and geometric considerations of the codon-anticodon base pairs to rank and to eliminate mRNA-tRNA pairs; the mechanism of fidelity check of the codon-anticodon pairing by the ribosome to evaluate noncanonical codon-anticodon base pairs and the role of water. We also consider certain tRNA modifications that interfere with the Ψ-coordinated water in the major groove of the codon-anticodon mini-helix. Our analysis of nonsense codons enables prediction of potential decoding properties for Ψ-modified sense codons, such as decoding ΨUU potentially as Cys and Tyr. Our results provide molecular rationale for the remarkable dynamics of ribosome decoding and insights on possible reprogramming of the genetic code using mRNA modifications.