Mutational alterations of tryptophan-specific transfer RNA that generate translation suppressors of the UAA, UAG and UGA nonsense codons

The recessive lethal amber suppressor su⁺7(UAG-1) in Escherichia coli inserts glutamine in response to the UAG codon. The genetic analysis presented in this paper shows that the su^?7 precursor allele can give rise to suppressors of the UGA codon as well as of the UAG codon. This observation suggests that the su^?7 gene normally codes for transfer RNA^Trp, a tRNA whose anticodon can be modified by single base changes to forms that can translate either UAG or UGA. The chemical findings presented in the accompanying paper (Yaniv et al., 1974) are wholly in accord with this interpretation. Thus, a single base substitution in the anticodon sequence of a tRNA can affect both the coding specificity of the molecule and also the amino acid acceptor specificity.     

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).     

Yeast UAA suppressors effective in psi+ strains: leucine-inserting suppressors.

We have previously reported the isolation and characterization of UAA suppressors from a haploid strain of yeast Saccharomyces cerevisiae containing the ψ⁺ non-Mendelian determinant which increases the efficiency of action of certain suppressors (Ono et al., 1979). Most of the suppressors caused the insertion of either tyrosine or serine. In contrast, the pattern of suppression of nutritional markers suggested that the rare suppressor, SUP26, inserted in an amino acid other than tyrosine or serine. In this investigation we report the characterization of additional suppressors, similar to SUP26, that were isolated on a medium lacking uracil and containing canavanine; this medium is expected to exclude serine-inserting suppressors because they do not suppress the ura4-1 marker, and to exclude tyrosine-inserting suppressors because they suppress the can1-100 marker. The total of 155 revertants similar to the SUP26 suppressor were analyzed genetically and these could be assigned to one or another of the six distinct loci SUP26, SUP27, SUP28, SUP29, SUP32 and SUP33. The SUP26, SUP27 and SUP29 loci mapped on chromosomes XII, IV and X, respectively. The detailed map position of the SUP29 suppressor suggests that it may be allelic to the SUP30 suppressor reported by Hawthorne &; Mortimer (1968). These six suppressors had the same pattern of suppression of UAA nutritional markers and all of them had a similar low efficiency of action on the iso-1-cytochrome c mutation cyc1-72. The efficiency of each of these suppressors was increased by a chromosomal allo-suppressor, sal. Each of the six suppressors caused the insertion of leucine in iso-1-cytochrome c at the UAA site of the cyc1-72 mutation. It is suggested that the gene products of these suppressors are redundant forms of the same leucine transfer RNA.     

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.     

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.     

mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG

Human mitochondria contain their own genome, encoding 13 polypeptides that are synthesized within the organelle. The molecular processes that govern and facilitate this mitochondrial translation remain unclear. Many key factors have yet to be characterized-for example, those required for translation termination. All other systems have two classes of release factors that either promote codon-specific hydrolysis of peptidyl-tRNA (class I) or lack specificity but stimulate the dissociation of class I factors from the ribosome (class II). One human mitochondrial protein has been previously identified in silico as a putative member of the class I release factors. Although we could not confirm the function of this factor, we report the identification of a different mitochondrial protein, mtRF1a, that is capable in vitro and in vivo of terminating translation at UAA/UAG codons. Further, mtRF1a depletion in HeLa cells led to compromised growth in galactose and increased production of reactive oxygen species.     

Dramatic events in ciliate evolution: alteration of UAA and UAG termination codons to glutamine codons due to anticodon mutations in two Tetrahymena tRNAs

The three major glutamine tRNAs of Tetrahymena thermophila were isolated and their nucleotide sequences determined by post-labeling techniques. Two of these tRNAs^Gln show unusual codon recognition: a previously isolated tRNA^Gln_UmUA and a second species with CUA in the anticodon (tRNA^Gln_CUA). These two tRNAs recognize two of the three termination codons on natural mRNAs in a reticulocyte system. tRNA^Gln_UmUA reads the UAA codon of α-globin mRNA and the UAG codon of tobacco mosaic virus (TMV) RNA, whereas tRNA^Gln_CUA recognizes only UAG. This indicates that Tetrahymena uses UAA and UAG as glutamine codons and that UGA may be the only functional termination codon. A notable feature of these two tRNAs^Gln is their unusually strong readthrough efficiency, e.g. purified tRNA^Gln_CUA achieves complete readthrough over the UAG stop codon of TMV RNA. The third major tRNA^Gln of Tetrahymena has a UmUG anticodon and presumably reads the two normal glutamine codons CAA and CAG. The sequence homology between tRNA^Gln_UmUG and tRNA^Gln_UmUA is 81%, whereas that between tRNA^Gln_CUA and tRNA^Gln_UmUA is 95%, indicating that the two unusual tRNAs^Gln evolved from the normal tRNA^Gln early in ciliate evolution. Possible events leading to an altered genetic code in ciliates are discussed.     

Interaction of UAG suppressors and omnipotent suppressors in Saccharomyces cerevisiae

Haploids bearing the dominant UAG suppressor, SUP7-a, and various alleles of the omnipotent suppressor sup35 were examined. The presence of the UAG suppressor reduced the efficiency of some alleles of sup35, and caused other sup35 alleles to be lethal. A nonclassical interaction of the dominant suppressor tRNA and the ribosome is proposed to explain these observations.     

Variations among glyV-derived glycine tRNA suppressors of glutamic acid codons.

Glutamic acid codon suppressors in 18 isogenic strains of Escherichia coli have been further characterized as to map location, dominance, growth rates in various media, suppression of the GAG codon, and tRNA profiles after reversed-phase column chromatography. In general the evidence supports the conclusion that all of these suppressors are due to mutations in glyV55, the gene for a GGA/G-reading mutant form of glyV tRNA, and that they represent several different classes that may correspond to at least as many different nucleotide changes. Furthermore, 17 of the 18 suppressors can coexist in a haploid genome with a glyT suppressor that is devoid of GGA-reading ability. This result indicates the retention by those glyV suppressors of some ability to respond to GGA as well as the acquisition of the ability to read GAA, and suggests the possibility of "wobble" in the middle position of the anticodons of those tRNA's.     

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.     

Modification of the anticodon triplet of E.coli tRNAMetf by replacement with trimers complementary to non-sense codons UAG and UAA.

E. coli tRNAMetf was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer. Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase. These modified tRNAMetf species had anticodons complementary to the termination codons UAG and UAA. Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer. Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAMetf accepted methionine. It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli. The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases.     

Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus: tRNA editing and UAG 'stop' codons recognized as leucine.

The mitochondrial DNA of the chytridiomycete fungus Spizellomyces punctatusen codes only eight tRNAs, although a minimal set of 24-25 tRNAs is normally found in fungi. One of these tRNAs has a CAU anticodon and is structurally related to leucine tRNAs, which would permit the translation of the UAG 'stop' codons that occur in most of its protein genes. The predicted structures of all S. punctatus tRNAs have the common feature of containing one to three mis-pairings in the first three positions of their acceptor stems. Such mis-pairing is expected to impair proper folding and processing of tRNAs from their precursors. Five of these eight RNAs were shown to be edited at the RNA level, in the 5'portion of the molecules. These changes include both pyrimidine to purine and A to G substitutions that restore normal pairing in the acceptor stem. Editing was not found at other positions of the tRNAs, or in the mitochondrial mRNAs of S. punctatus. While tRNA editing has not been observed in other fungi, the editing pattern inS.punctatus is virtually identical to that described in the amoeboid protozoan Acanthamoeba castellanii. If this type of mitochondrial tRNA editing has originated from their common ancestor, one has to assume that it was independently lost in plants, animals and in most fungi. Alternatively, editing might have evolved independently, or the genes coding for the components of the editing machinery were laterally transferred.     

Missense and nonsense suppressors derived from a glycine tRNA by nucleotide insertion and deletion in vivo

Summary Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia coli and each containing an extra nucleotide in the anticodon loop, we generated new suppressors in vivo by spontaneous deletion of specific nucleotides from the anticodon loop. In one experiment, the new suppressor was generated by a double mutational event, base substitution and nucleotide deletion. A novel ochre suppressor is also described. It is very efficient in nonsense suppression but has no ms²i⁶ modification of the A residue on the 3 side of the anticodon. The results have important implications for tRNA structure-function relationships, tRNA recognition by tRNA-modifying enzymes, mechanisms of deletion mutation, and tRNA evolution.A preliminary report of these results was presented at the EMBO Workshop on Accuracy, Grignon, France, September 1–6, 1981