Nucleotide alterations in the bacteriophage T4 glutamine transfer RNA that affect ochre suppressor activity

We have determined the nucleotide sequences of the glutamine transfer RNAs that are coded by wild-type and psu₂⁺ ochre-suppressor strains of bacteriophage T4. The two transfer RNAs have the same sequence except for their anticodons, where NUG in the wild-type species is mutated to NUA in the psu₂⁺ species (N is a modified residue of U). This mutation is believed to confer suppressor activity on the psu₂⁺ glutamine tRNA. Three mutants derived from psu₂⁺ by loss of suppressor activity have been characterized with respect to their sequence alterations. Each mutant specifies a transfer RNA differing from the psu₂⁺ species by a nucleotide substitution that occupies a base-paired region in the cloverleaf arrangement of the molecule. The mutants synthesize a reduced amount of tRNA that is defective in nucleotide modifications and processing at the 5′ and 3′ termini.     

Characterization of an amber suppressor in Pneumococcus.

Summary Partial revertant has been isolated, with resistance to aminopretin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su⁺) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su⁺ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidilate synthetase gene are suppressible. Thymidilate synthetase activity is partially restored by su⁺. Optochin mutants can also be suppressed. Thus su⁺ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycine is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor.The t-RNA extracted from the suppressor strain su⁺ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su⁺ gene is amber specific.Thus su⁺ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su⁺; this is additional evidence that both categories contain point mutations.     

Identification of transfer RNA suppressors in Escherichia coli. I. Amber suppressor su+2, an anticodon mutant of tRNA2Gln.

In order to isolate the gene for amber suppressor su⁺2 (SupE) in Escherichia coli, a non-defective su⁺2-transducing phage lambda was isolated in three steps: first, deletion derivatives of F′su⁺2 gal (λ) were selected, linking su⁺2 to the right-hand prophage attachment site, att^λPB′; second, these F′-factors were relysogenized by λ and defective transducing phages, λdsu⁺2, were produced by induction; and third, non-defective λpsu⁺2 transducing phages were produced by recombination of λdsu⁺2 isolates with λ. Upon infection by λpsu⁺2, the production of transferRNAs accepting glutamine and methionine was markedly stimulated. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNA₂^Gln, mutant tRNA₂^Gln and tRNA_m^Met. The mutant tRNA₂^Gln carried a singlebase alteration from G to A at the 3′-end of the anticodon. The production of tRNA₁^Gln was not stimulated by the infection of λpsu⁺2. We conclude that the wild-type allele of su⁺2 (SupE) is the structural gene for tRNA₂^Gln, and the su⁺2 amber suppressor was derived by a single base mutation, changing the anticodon from CUG to CUA, in one of the multi-copy genes for tRNA₂^Gln. The fact that λpsu⁺2 also induces the production of tRNA_m^Met suggests that this tRNA is encoded in the same chromosomal region of E. coli as is tRNA₂^Gln.     

Identification of transfer RNA suppressors in Escherichia coli. II. Duplicate genes for tRNA2Gln.

The number of gene copies for tRNA₂^Gln in λpsu⁺2 was determined by genetic and biochemical studies. The transducing phage stimulates the production of the su⁺2 (amber suppressor) and su°2 glutamine tRNAs and methionine tRNA_m. When the su⁺2 amber suppressor was converted to an ochre suppressor by single-base mutation, the phage stimulated ochre-suppressing tRNA₂^Gln, instead of the amber-suppressing tRNA₂^Gln. From the transducing phage carrying the ochre-suppressing allele, strains carrying both ochre and amber suppressors were readily obtainable. These phages stimulated both ochre-suppressing and amber-suppressing tRNA₂^Gln, but not the non-suppressing form. We conclude that the original transducing phage carries two tRNA₂^Gln genes, one su⁺2 and one su°2. The transducing phage carrying two suppressors, ochre and amber, segregates one-gene derivatives that encode only one or the other type of suppressor tRNA. These derivatives apparently arise by unequal recombination involving the two glutamine tRNA genes in the parental phage. This segregation is not accompanied by the loss of the tRNA_m^Met gene. Based on these results, it is suggested that Escherichia coli normally carries in tandem two identical genes specifying tRNA₂^Gln at 15 minutes on the bacterial chromosome. su⁺2 mutants may arise by single-base mutations in the anticodon region of either of these two, leaving the other intact. By double mutations, tRNA₂^Gln genes could also become ochre suppressors. A tRNA_m^Met gene is located near, but not between, these two tRNA₂^Gln genes.     

Purification of a hybrid molecule composed of tyrosine transfer RNA and its complementary DNA sequence

The transducing bacteriophage φ80psu_III⁺ carries one structural Escherichia coli gene specifying tyrosine tRNA.The r strand of bacteriophage φ80psu_III⁺ was hybridized with E. coli transfer RNA and the hybrid digested with Neurospora crassa endonuclease. The analysis of the products of enzymic digestion demonstrated the release of a cistron-hybrid composed of tyrosine tRNA and its complementary DNA sequence. The cistron-hybrid was purified from unhybridized DNA by cesium sulphate density-gradient centrifugation and gel filtration.The ratio between tyrosine tRNA and its complementary DNA sequence in the final product was 1:1 as demonstrated by radioisotopic analysis. This purification represents a 30,000-fold enrichment of the E. coli genome for a specific DNA sequence.     

Letter: Mutant tyrosine transfer ribonucleic acids of Escherichia coli: construction by recombination of a double mutant A1G82 chargeable with glutamine

A double mutant A1G82 of the su_III⁺ tyrosine gene in Escherichia coli was constructed by genetic cross. This mutant is a stronger glutamine-inserting amber suppressor than either of the single mutants A1 or G82.     

Isolation of an Escherichia coli strain restricting bacteriophage suppressor

Summary A bacterial mutant is described which restricts bacteriophage amber suppressor psu ⁺. Restriction, probably, operates at a translational level. The new strain provides the system of identification of bacteriophage amber mutants suppressed by psu ⁺ using suppressornegative (su ^-) bacterial strains.     

Yeast suppressors of UAA and UAG nonsense codons work efficiently in vitro via tRNA

A cell-free protein-synthesizing system, containing an S-100 fraction from yeast, ribosomal subunits from Krebs ascites cells, and ribosome initiation factors from rabbit reticulocytes, translates yeast, adenovirus, and rabbit globin messenger RNAs and the RNA from bacteriophage Qβ. An amber mutation in the Qβ synthetase gene is suppressed in vitro if the S-100 fraction is from yeast strains carrying amber suppressor mutations. Suppressor SUP6-2 gives 16% suppression, and the recessive lethal suppressor RL-1 gives 50% suppression. Extracts from strain FM6, which has the ochre suppressor SUP4-1, give a longer protein product from the normal synthetase gene of Qβ with an efficiency of 63%. This implies that UAA is the terminator for the synthetase gene, and that synthesis of this read through protein can be used as an assay for ochre suppression. Suppression in each of these cases is mediated by tRNA, since purified tRNA is the only fraction from suppressing strains that is required in an otherwise nonsuppressing cell-free system.     

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.     

Isolation and characterization of a temperature-sensitive amber suppressor mutant of Escherichia coli K12

Summary By mutagenizing an E. coli strain carrying an amber suppressor supD ^- (or su _I ⁺), we isolated a mutant whose amber suppressor activity was now temperature-sensitive. The mutant suppressor gene was named sup-126, which was found to be cotransduced with the his gene by phage P1vir at the frequency of ca. 20%. At 30° C it suppresses many amber mutations of E. coli, phage T4, and phage . At 42° C, however, it can suppress none of over 30 amber mutations tested so far. The sup-126 mutation is unambiguous and stable enough to be useful for making production of an amber protein temperature-sensitive.     

Nanovariant RNAs: nucleotide sequence and interaction with bacteriophage Qbeta replicase

Gene 2 amber mutants of bacteriophage T4 grown on su^? hosts produce whole particles of which less than 0.5% are infective on su⁺ hosts. Although the DNA of such particles is full-sized and un-nicked, it is degraded to acid-soluble fragments after infection of exo V⁺ hosts. This breakdown does not occur on exo V^? deficient hosts, and such hosts are fully permissive for gene 2-defective particles. We have now determined that giant-headed, gene 2-defective particles containing several genome lengths of DNA per head are fully infective on exo V⁺ hosts even though part of the parental DNA is degraded to acid-soluble fragments early after infection. Restriction of gene 2-defective particles must therefore be due to exonucleolytic degradation of the incoming DNA. If the parental DNA is of sufficient length to enable a complete genome to survive this degradation before production of anti-exoV, such particles are now infective.     

Dual specificity of su+7 tRNA. Evidence for translational discrimination

The su⁺7 amber suppressor of Escherichia coli is a mutant tRNA^Trp that translates UAG codons as glutamine. Nevertheless, the purified su⁺7 tRNA can be charged with either glutamine or tryptophan. Aminoacylation kinetics in vitro suggest that the tRNA should be acylated with equal amounts of glutamine and tryptophan in vivo. The predominance of the glutamine specificity of the suppressor is therefore potentially anomalous. We can find no selective deacylation of tryptophanyl-su⁺7 tRNA by glutaminyl-tRNA synthetase, tryptophanyl-tRNA synthetase, or any other cellular element. Furthermore, as predicted, nearly equal amounts of glutaminyl and tryptophanyl-su⁺7 tRNA are actually detected in aminoacyl-tRNA extracted from growing cells. We conclude that the translational apparatus somehow discriminates against tryptophanyl-su⁺7 tRNA at a step after synthesis of the two aminoacyl-tRNAs.     

A single mutational modification of a tryptophan-specific transfer RNA permits aminoacylation by glutamine and translation of the codon UAG

In this work we show that the wild-type (su^?7) progenitor of the recessivelethal suppressors of UAG (su⁺7(UAG)) and of UAA/G (su⁺7(UAA/G)) is the structural gene for transfer RNA^Trp, the adaptor for translating the codon UGG. The su⁺7(UAG) suppressor form of the tRNA has a C for U substitution in the middle base of the anticodon; in the su⁺7(UAA/G) suppressor tRNA both C residues of the anticodon are replaced by U. Our data establish that the mutational change altering the tRNA^Trp to a UAG suppressor is accompanied by a loss of tryptophan-accepting specificity and the acquisition of glutamine-acceptor activity.     

Relative stabilities of RNA/DNA hybrids: effect of RNA chain length in competitive hybrization

Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA^Tyr-like sequences transcribed in vitro on φ80psu₃⁺ bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA^Tyr hybridization revealed that the in vitro-synthesized RNA contained significant amounts of tRNA^Tyr; these sequences could not, however, be detected by the direct competition method in which labelled in vitro-synthesized RNA competes with E. coli tRNA^Tyr for hybridization to φ80psu₃⁺ DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro-transcribed tRNA^Tyr-like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA^Tyr from its hybrids with φ80psu₃⁺ DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA^Tyr.     

Mutants of satellite bacteriophage P4 that are defective in the suppression of transcriptional polarity

The satellite bacteriophage P4 relies on a helper such as P2 to supply the gene products necessary for virion assembly and cell lysis (Six, 1975). P4 has the unique capacity to activate the late genes of P2 by a mechanism that differs from the one normally used by P2 itself. This process has been termed transactivation (Calendar et al., 1977). In addition, P4 is able to suppress the strong polarity associated with certain P2 amber mutations. The isolation of P4 mutants solely defective in polarity suppression (psu^?) demonstrates that the ability of P4 to suppress polarity is non-essential for P4 growth. In particular, polarity suppression plays no essential role in either transactivation or head size determination. The product of the P4 psu gene has been identified as a 19,900 M_r P4 late protein.     

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

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

Mutation of bacteriophage T4 restoring phage supressor psul+ activity in bacterial strain Escherichia coli BN]

The mutant of bacteriophage T4psu1+XF2 carrying a mutational aleration in the central region of proline-serine tRNA precursor is isolated. The mutational alteration results in the recovery of amber suppressor activity of phage psu1+ serine tRNA in Escherichia coli BN in which the synthesis of this tRNA is normally blocked. Since the amber suppressor activity of mutant serine tRNA becomes sensitive to a restrictive action of strR mutations, its structure seems to be different from that of parental suppressor serine tRNA.     

Bacteriophage T4 transfer RNA. I. Isolation and characterization of two-phage-coded nonsense suppressors

Two phage-coded nonsense suppressors, psuf_a⁺ and psu_b⁺, have been isolated and characterized. Both were isolated as pseudo-wild type revertants of phage strains which carry multiple amber mutations. psu_a⁺ is an amber suppressor which occurs at a frequency of 10⁻¹¹ to 10⁻¹² and is indistinguishable from wild type phage in its growth on both B and K strains of Escherichia coli bacteria. psu_b⁺ may be either an amber or an ochre suppressor, which occurs at a frequency of 10⁻⁷ to 10⁻¹⁰ and makes small plaques on B strains, but grows very poorly or not at all on K strains. Phage with the characteristics of psu_a⁺ occur in populations of psu_b⁺ phage at a frequency of 10⁻⁴. Both suppressors insert serine in response to the amber codon at an efficiency of about 45%.