A double base change in alternate base pairs induced by ultraviolet irradiation in a glycine transfer RNA gene

Summary The glyUsu _AGA mutation affects Escherichia coli tRNA _GGG ^{G1 y} , changing it to an AGA missense suppressor tRNA. Sequence studies have shown that the mutation involves a double base substitution at the first and third positions of the tRNA anticodon, the result being a change in the anticodon from CCC to UCU. A system has been developed to facilitate the detection of this novel mutation, and we have shown that ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are effective in causing the double base change. A single observation of the mutation occurring spontaneously has been made also. The frequency of MNNG-induced glyUsu _AGA mutations is compatible with their being caused by two separate mutagenic events. The frequency of UV-induced glyUsu _AGA mutations, however, strongly suggests that the occurrence of one base substitution strongly enhances the chance of finding the second substitution at the alternate position.In addition to the double change in the anticodon, the glyUsu _AGA tRNA differs from tRNA _GGG ^{G1 y} in that it bears a modification of the A adjacent to the 3 position of the anticodon. Most likely, this modified base is N-[9-(-D-ribofuranosyl)-purin-6-ylcarbamoyl] threonine.     

Three Different Missense Suppressor Mutations Affecting the tRNAGGGGly Species of Escherichia coli

The Escherichia coli suppressor mutation, supT, has been shown to cause a C → U substitution in the middle position of the tRNA_GGG^Gly anticodon. This is the same tRNA species that is altered by the glyUsu_AGA mutation studied previously. This finding indicates that the supT mutant tRNA reads the glutamic acid codon, GAG. The supT suppressor has also been converted to a new suppressor, called glyUsu_GAA, which will suppress the GAA mutation, trpA46. The in vivo suppression efficiencies of each of these three missense suppressors has been measured and are as follows: glyUsu_AGA, 3.6%; supT, 1.6%; and glyUsu_GAA, 0.4%. Mistranslation by these mutant glycine tRNA species has no adverse affects on cell growth since cultures possessing the suppressors grow as fast as cells without. The supT tRNA species can be observed as a peak in the profile of glycyl-tRNA fractionated on a RPC-5 chromatographic column, indicating that the mutant tRNA can be aminoacylated with reasonable efficiency. This finding contrasts with previous findings concerning the glyUsu_AGA mutant tRNA which is not significantly aminoacylated under the same conditions.     

Mutant of the glutamine transfer RNA gene as UGA suppressor in Escherichia coli

Summary A UGA suppressor derived from a glutamine tRNA gene of Escherichia coli K 12 was isolated and characterized. Phages carrying the suppressor su⁺2_UGA could be obtained only from a hybrid transducing phage, h ⁸⁰ cI ₈₅₇psu ⁺2_oc, but not from the original transducing phage cI ₈₅₇psu ⁺2_oc. By DNA sequence analysis, it was found that the su ⁺2 UGA suppressor obtained has two mutations; one is in the anticodon (TTATCA), as expected, and the other (CT) is at the 7th position from the 3 end of tRNA ₂ ^Gln . The significance of these mutations and the lethal effect on phage of the increased amounts of UGA suppressor tRNAs are discussed.     

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.     

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     

Identification and nucleotide sequence of the sup8-e UGA-suppressor leucine tRNA from Schizosaccharomyces pombe.

Summary Using the translation of rabbit globin mRNA in wheat germ extracts as an assay for ochre and opal suppression, a UGA suppressor tRNA from Schizosaccharomyces pombe strain sup8-e was purified by column chromatography and two-dimensional gel electrophoresis. The purified tRNA can be aminoacylated with leucine by a crude aminoacyl-tRNA synthetase preparation from a wild type S. pombe strain, and has high activity in the suppressor assay. By a combination of post-labeling fingerprinting and rapid gel sequencing methods the nucleotide sequence of this suppressor tRNA was determined to be: pG-C-G-G-C-U-A-U-G-C-C-ac⁴C-G-A-G-D-G-D-G-D-A-A-G-G-G-m ₂ ² G-G-C-A-G-A--U-U^*-C-A-m¹G-C-C-C-U-G-C-U-G-U-U-G-U-A-A-A-A-C-G-m⁵C-G-A-G-A-G-T--C-G-m¹A-A-C-C-U-C-U-C-U-G-G-C-C-G-C-A-C-C-A_OH. The anticodon sequence U^*CA is complementary to the UGA codon. An interesting feature of the suppressor tRNA is an expanded anticodon loop of nine nucleotides owing to an A-C nonpair at the first anticodon stem position.     

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.     

Effect of DNA sequence changes on UV mutability of a purine anticodon triplet of glyU cloned on M13 phage

Summary Mutant forms of the glyU (glycyl tRNA) gene cloned in M13mp8 were subjected to uninduced targeted UV mutagenesis; i.e. phage particles were irradiated and used to infect unirradiated umuC ⁺ or irradiated umuC mutant cells. The irradiated phage carried GAG at the anticodon triplet and transitions to GAA were scored. The uninduced targeted mutation rate was reduced by altering the sequence of the gene in the vicinity of the target purine (Pu) residue. In particular a triplet of pyrimidines (PyPyPy) 5 to the target G was changed to PyPuPy in order to prevent formation of cyclcobutane and 6-4 pyrimidine dimers close to the target. On this basis we suggest a mechanism for one type of uninduced regionally targeted UV mutagenesis.     

Analysis of ultraviolet light-induced suppressor mutations in the strain of Escherichia coli K-12 AB1157: an implication for molecular mechanisms of UV mutagenesis

Summary Genetic analysis of histidine independent (His⁴) revertants induced by ultraviolet light in the his-4 E. coli strain AB1157 was carried out: 83% carried ochre (UAA) suppressor mutations and 17% carried back mutations to his ⁺ or (intragenic?) suppressors not detectably separable from his-4. Using the specialized transducing psu 2int ^– phage, which carries supE-supB, it was determined that 87% of the ochre suppressors mapped in the supE-supB region. We were able to deduce that 56% of these affected tRNA ₁ ^Gln by a CAATAA change in the tRNA gene while 31% affected tRNA ₂ ^Gln by TAGTAA change. Although we were unable to deduce the base substitution of the remaining 13%, the results indicated that most of the suppressor mutations are caused by a G:C to A:T transition.These results suggest that the high incidence of supE-supB region suppressor mutation in E. coli by UV would be a reflection of the general feature of UV mutagenesis; i.e. preferential induction of G:C to A:T transition in repairing nonparing DNA lesions.     

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.     

Normal and Mutant Glycine Transfer RNAs

THE glycine-specific tRNAs of E. coli can be grouped into three subspecies which are separated by chromatography on benzoylated DEAE cellulose (BDC): tRNA^Gly₁ (GGG), tRNA^Gly₂ (GGA/G) and tRNA^Gly₃ (GGU/C)^1,2. The tRNA^Gly₁ and tRNA^Gly₂ are specified by the genes, glyU and glyT, respectively, which have been located at 55 and 77 minutes on the E. coli chromosome. Suppressors of tryptophan A gene (trpA) missense mutations and partial diploid strains have been used extensively to characterize the glycine tRNA structural genes (Table 1)^1–3. A common property of these suppressor mutations is that the altered tRNA^Gly is no longer aminoacylated at the normal rate by the glycyl tRNA synthetase (GRS). When ordinary loading conditions are used virtually none of the suppressor tRNA species are amino-acylated. These studies have shown that single gene copies are normally present at the glyT and glyU loci.     

Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA^Tyr _{G A} and elongator tRNA^Met _CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA^Tyr. Here we have studied the expression of an Arabidopsis elongator tRNA^Met gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA^Met precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA^Met to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3 and 5 splice sites and of a structured intron for pre-tRNA^Met splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA^Met splicing and that a highly structured intron is indispensable for pre-tRNA^Met splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA^Met gene, is efficiently processed and spliced in both plant extracts.     

Conformational dynamics of the anticodon loop in yeast tRNAPhe as sensed by the fluorescence of wybutine

Conformational and dynamic properties of the anticodon loop of yeast tRNA^Phe were investigated by analyzing the time resolved fluorescence of wybutine serving as a local structural probe adjacent to the anticodon GmAA on its 3 side. The influence of Mg²⁺, important for stabilizing the tertiary structure of tRNA, and of the complementary anticodon s²UUC of E. coli tRNA ₂ ^Glu were investigated.Fluorescence lifetimes and anisotropies were measured with ps time resolution using time correlated single photon counting and a mode locked synchronously pumped and frequency doubled dye laser as excitation source. From the analysis of lifetimes () and rotational relaxation times ( _R ) we conclude that wybutine occurs in various structural states: (i) one stacked conformation where the base has no free mobility and the only rotational motion reflects the mobility of the whole tRNA molecule (=6 ns, _R =19 ns), (ii) an unstacked conformation where the base can freely rotate (=100 ps, _R = 370 ps) and (iii) an intermediary state (=2 ns, _R = 1.6 ns).Under biological conditions, i. e. in the presence of Mg²⁺ and neutral salts, wybutine is found in a stacked and immobile state which is consistent with the crystallographic picture. In the presence of the complementary codon however, as exemplified by the E. coli-tRNA ₂ ^Glu anticodon, our analysis indicates that the codon-anticodon complex exists in an equilibrium of structural states with different rotational mobility of wybutine. The conformation with wybutine freely mobile is the predominant one and suggests that this conformation of the codon-anticodon structure differs from the canonical 3–5 stack.     

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.     

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.     

Interstrain crosses enhance excision of Tc1 transposable elements in Caenorhabditis elegans

Summary We have studied spontaneous and UV mutagenesis of the glyU gene in Escherichia coli trpA461 (GAG) strains carrying the pIP11 plasmid, in which the dnaQ gene encoding the 3–5 exonuclease subunit (epsilon) of DNA polymerase III is fused to the tac(trp-lac) promoter. We have used a pair of M13glyU phage in which the gene encoding the glycyl-tRNA is cloned in opposite orientations, consequently the phage present either GGG or CCC anticodon triplets for mutagenesis. The presence of IPTG, the inducer of the tac-dnaQ fusion, results in about 100-fold decrease in frequency of spontaneous Su⁺ (GAG) mutations arising in the CCC phage. The enhanced expression of tac-dnaQ reduces 10-fold the frequency of UV-induced Su⁺ (GAG) mutations in the CCC phage and nearly completely prevents generation by UV of Su⁺ (GAG) mutations in the GGG phage, in which UV-induced pyrimidine photoproducts can be formed only in the vicinity of the target triplet. These results suggest that both locally and regionally targeted mutagenesis is affected by overproduction of the epsilon subunit. By delayed photoreversal mutagenesis we have shown that UV-induced chromosomal mutagenesis of the umuC36 trpA461 strain harboring pIP11 is completely abolished in the presence of IPTG. This result seems to indicate that the misinocorporation step of DNA translesion synthesis is affected by excess of the epsilon subunit. Finally, we have introduced the pIP13 plasmid carrying the dnaQ gene into the recA1207 strain, which is deficient in the recombinase activity of RecA but constitutive in the protease activity. We demonstrate that the transformant shows much higher UV sensitivity than recA1207 carrying the vector plasmid pBR325, indicating that translesion synthesis significantly contributes to DNA repair capacity of cells deficient in recombination.     

Nonsense mutants of Serratia phage Kappa

Summary Auxotrophs of Serr. marcescens HY, which behaved like nonsense mutants when tested according to Whitfield et al. (1966), were induced to revert to anauxotrophy. Some of the revertants (called su ⁺), together with the parental auxotrophs (called su ^-), allowed to isolate conditional-lethal (sus) mutants of phage Kappa, which produce infectious progeny only in su ⁺ bacteria. All su ⁺ mutants of Serr. marcescens HY were identified as nonsense suppressors using su ⁺ _amber, su ⁺ _ochre, and su ^- strains of Salm. typhimurium as references and the flagella-specific phage Chi as the main tool to connect the Salmonella system with that of Serratia.After treatment of Kappa with three different mutagens 128 sus mutants were isolated which comprise at least 19 complementation groups. 18 sus mutants, representing different cistrons, and the unselective markers c1, c2, and c49 were mapped mainly by two-factor crosses. Reciprocal three-factor crosses of the general type a x bz and az x b (i.e. with outside markers) revealed a circular linkage map of an estimated maximum length of 90 RU (recombination units). Joined rescue of outside markers, e.g. sus ⁺ A94 and e49, from UV-irradiated phage supported the assumption of circular gene linkage. Some data indicate that certain regions of the phage genome might have a higher chance to recombine than others.Abbreviations moi multiplicity of infection - eop efficiency of plating - RU recombination units - MR marker rescue