Suppression of Glutamic Acid Codons by Mutant Glycine Transfer Ribonucleic Acid

In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.     

Genetic alteration of structure and function in glycine transfer RNA of Escherichia coli: Mechanism of suppression of the tryptophan synthetase A78 mutation

Suppression (su_A78⁺) of the trpA78 missense mutation (Gly,GGU/C → Cys,UGU/C) in Escherichia coli involves a genetically altered tRNA^Gly isoacceptor. Purification and sequence analysis of ³²P-labeled suppressor tRNA indicates that the su_A78⁺ mutation results in the alteration of a small fraction of the tRNA_GGU/C^Gly3 of the cell. The resulting tRNA_UGU/C^Gly3 contains a C → A substitution at the 3′ end (C36) of the anticodon (GCC) and in addition, a modification of the adjacent A(A37) to form N⁶-(Δ²-isopentenyl)-2-thiomethyl-adenylic acid. Labeled glycyl-tRNA_UGU/C^Gly3 binds to E. coli ribosomes in the presence of the cysteine triplets, UpGpU and UpGpC, but not with the glycine triplets, GpGpU and GpGpC. The suppressor tRNA_UGU/C^Gly3 glycylates relatively slowly in the presence of the glycyl-tRNA synthetase, with a V_max value 200-fold lower than that of wild-type tRNA_GGU/C^Gly3. Multiple identical copies of genes specifying the sequence of tRNA_GGU/C^Gly3 apparently occur on the E. coli chromosome, since suppressor mutations alter the nucleotide sequence of only a fraction of the tRNA_GGU/C^Gly3 population.     

Specificity of spontaneous mutations induced in mutA mutator cells

Escherichia coli cells expressing the mutA allele of a glyV (glycine tRNA) gene express a strong mutator phenotype. The mutA allele differs from the wild type glyV gene by a base substitution in the anticodon such that the resulting tRNA misreads certain aspartate codons as glycine, resulting in random, low-level Asp-->Gly substitutions in proteins. Subsequent work showed that many types of mistranslation can lead to a very similar phenotype, named TSM for translational stress-induced mutagenesis. Here, we have determined the specificity of forward mutations occurring in the lacI gene in mutA cells as well as in wild type cells. Our results show that in comparison to wild type cells, base substitutions are elevated 23-fold in mutA cells, as against a eight-fold increase in insertions and a five-fold increase in deletions. Among base substitutions, transitions are elevated 13-fold, with both G:C-->A:T and A:T-->G:C mutations showing roughly similar increases. Transversions are elevated 35-fold, with G:C-->T:A, G:C-->C:G and A:T-->C:G elevated 28-, 13- and 27-fold, respectively. A:T-->T:A mutations increase a striking 348-fold over parental cells, with most occurring at two hotspot sequences that share the G:C-rich sequence 5'-CCGCGTGG. The increase in transversion mutations is similar to that observed in cells defective for dnaQ, the gene encoding the proofreading function of DNA polymerase III. In particular, the relative proportions and sites of occurrence of A:T-->T:A transversions are similar in mutA and mutD5 (an allele of dnaQ) cells. Interestingly, transversions are also the predominant base substitutions induced in dnaE173 cells in which a missense mutation in the alpha subunit of polymerase III abolishes proofreading without affecting the 3'-->5' exonuclease activity of the epsilon subunit.     

Structural Interactions Between Amino Acid Residues at Positions 22 and 211 in the Tryptophan Synthetase Alpha Chain of Escherichia coli

Construction and characterization of double mutants altered in the structural gene of the tryptophan synthetase alpha chain of Escherichia coli revealed interactions between amino acid residues at positions 22 and 211. These interactions are specific for the particular amino acid residue at position 211. The results indicate also that amino acid residues which appear to be functionally near-equivalent in one configuration may strongly influence the activity of a protein with a subsequent change at another site. Seven independent suppressors of trpA218 (Leu22-Ser211) were isolated. Their properties suggest that all seven may suppress the codon (AGU/C) for Ser211. Six of the seven are co-transducible with glyV, the structural gene for the GGU/C-specific tRNA(Gly).     

Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells.

Translational stress-induced mutagenesis (TSM) refers to the mutator phenotype observed in Escherichia coli cells expressing a mutant allele (mutA or mutC) of the glycine tRNA gene glyV (or glyW). Because of an anticodon mutation, expression of the mutA allele results in low levels of Asp-->Gly mistranslation. The mutA phenotype does not require lexA-regulated SOS mutagenesis functions, and appears to be suppressed in cells defective for RecABC-dependent homologous recombination functions. To test the hypothesis that the TSM response is mediated by non-specific mistranslation rather than specific Asp-->Gly misreading, we asked if streptomycin (Str), an aminoglycoside antibiotic known to promote mistranslation, can provoke a mutator phenotype. We report that Str induces a strong mutator phenotype in cells bearing certain alleles of rpsL, the gene encoding S12, an essential component of the ribosomal 30 S subunit. The phenotype is strikingly similar to that observed in mutA cells in its mutational specificity, as well as in its requirement for RecABC-mediated homologous recombination functions. Expression of Str-inducible mutator phenotype correlates with mistranslation efficiency in response to Str. Thus, mistranslation in general is able to induce the TSM response. The Str-inducible mutator phenotype described here defines a new functional class of rpsL alleles, and raises interesting questions on the mechanism of action of Str, and on bacterial response to antibiotic stress.     

Nucleotide sequence studies of normal and genetically altered glycine transfer ribonucleic acids from Escherichia coli.

The total nucleotide sequence of tRNAGGA/G -Gly2 from Escherichia coli is pG-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-C-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-AOH, where T- at position 53 is ribothymidylic acid, and psi- at position 54 is pseudouridylic acid; N- at position 36 is an unidentified derivative of uridylic acid, and is present in modified form in a portion of tRNAGGA/G -Gly 2 molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNAGGA/G -Gly 2 (nucleotide position 38). A secondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNAsuA36(HA), -Gly 2 suggesting that the enzymes responsible for this modification recognize the anticodon sequences of prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNAsuA36(HA), -Gly 2 by an alteration of the anticodon sequence of tRNAGGA/G -Gly 2 is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions of four glycine isoaccepting tRNAs specified by E. coli and bacteriophage T4 suggests that these regions may be recognized by the glycyl-tRNA synthetase; the involvement of the anticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNAsuA36(HA) -Gly 2.     

Mutations which alter the elbow region of tRNA2Gly reduce T4 gene 60 translational bypassing efficiency.

Translating ribosomes bypass a 50 nucleotide coding gap in bacteriophage T4 gene 60 mRNA between codons 46 and 47 in order to synthesize the full-length protein. Bypassing of the coding gap requires peptidyl-tRNA2Gly detachment from a GGA codon (codon 46) followed by re-pairing at a matching GGA codon just before codon 47. Using negative selection, based on the sacB gene from Bacillus subtilis, Escherichia coli mutants were isolated which reduce bypassing efficiency. All of the mutations are in the gene for tRNA2Gly. Most of the mutations disrupt the hydrogen bonding interactions between the D- and T-loops (G18*psi55 and G19*C56) which stabilize the elbow region in nearly all tRNAs. The lone mutation not in the elbow region destabilizes the anticodon stem at position 40. Previously described Salmonella typhimurium mutants of tRNA2Gly, which reduce the stability of the T-loop, were also tested and found to decrease bypassing efficiency. Each tRNA2Gly mutant is functional in translation (tRNA2Gly is essential), but has a decoding efficiency 10- to 20-fold lower than wild-type. This suggests that rigidity of the elbow region and the anticodon stem is critical for both codon-anticodon stability and bypassing.     

Frameshift suppressor mutations affecting the major glycine transfer RNAs of Saccharomyces cerevisiae

Mutations have been identified in Saccharomyces cerevisiae glycine tRNA genes that result in suppression of +1 frameshift mutations in glycine codons. Wild-type and suppressor alleles of genes encoding the two major glycine tRNAs, tRNA(GCC) and tRNA(UCC), were examined in this study. The genes were identified by genetic complementation and by hybridization to a yeast genomic library using purified tRNA probes. tRNA(UCC) is encoded by three genes, whereas approximately 15 genes encode tRNA(GCC). The frameshift suppressor genes suf1+, suf4+ and suf6+ were shown to encode the wild-type tRNA(UCC) tRNA. The suf1+ and suf4+ genes were identical in DNA sequence, whereas the suf6+ gene, whose DNA sequence was not determined, was shown by a hybridization experiment to encode tRNA(UCC). The ultraviolet light-induced SU F1-1 and spontaneous SU F4-1 suppressor mutations were each shown to differ from wild-type at two positions in the anticodon, including a +1 base-pair insertion and a base-pair substitution. These changes resulted in a CCCC four-base anticodon rather than the CCU three-base anticodon found in wild-type. The RNA sequence of tRNA(UCC) was shown to contain a modified uridine in the wobble position. Mutant tRNA(CCCC) isolated from a SU F1-1 strain lacked this modification. Three unlinked genes that encode wild-type tRNA(GCC), suf20+, trn2, and suf17+, were identical in DNA sequence to the previously described suf16+ frameshift suppressor gene. Spontaneous suppressor mutations at the SU F20 and SU F17 loci were analyzed. The SU F20-2 suppressor allele contained a CCCC anticodon. This allele was derived in two serial selections through two independent mutational events, a +1 base insertion and a base substitution in the anticodon. Presumably, the original suppressor allele, SU F20-1, contained the single base insertion. The SU F17-1 suppressor allele also contained a CCCC anticodon resulting from two mutations, a +1 insertion and a base substitution. However, this allele contained an additional base substitution at position 33 adjacent to the 5' side of the four-base anticodon. The possible origin and significance of multiple mutations leading to frameshift suppression is discussed.     

Increased levels of glycine tRNA associated with collagen synthesis

Analysis of codon usage for chick Type I collagen indicates that 89% of glycine codons are GGU/C. Since collagens are one-third glycine, chick Type I collagen synthesis should require large amounts of tRNAGly with the anticodon GCC. Earlier chromatographic studies of chick tRNA had indicated that connective tissues showed altered tRNAGly isoacceptor profiles [P. J. Christner and J. Rosenbloom (1976) Arch. Biochem. Biophys. 172, 399-409; H. J. Drabkin and L. N. Lukens (1978) J. Biol. Chem. 253, 6233-6241]. We have therefore used both two-dimensional gel electrophoresis and hybridization analysis to investigate whether collagen synthesis in chick connective tissues is associated with expression of a novel tRNAGly. Liver and calvaria tRNAs produced qualitatively similar patterns when separated on 2-D gels. Northern blots of 2-D-separated tRNAs from liver and calvaria, when hybridized to genes for vertebrate tRNAGly isoacceptors with GCC or UCC anticodons, showed hybridization to the same tRNAs in both tissues. Quantitation of tRNA species by dot blot hybridization indicated an increase in levels of the tRNAGly isoacceptor with anticodon GCC. Tissues synthesizing Type I collagen had a two- to threefold increase in this tRNA while tissues synthesizing Type II collagen showed a more modest increase. We conclude that elevated tRNAGly levels associated with collagen synthesis are due to increased amounts of the same isoacceptor which is the major tRNAGly in other tissues.     

Reaction heat variation with pH in formation of the trypsin-soybean inhibitor complex.

The heat of reaction between beta-trypsin and Kunitz soybean inhibitor (STI) hasbeen measured at 5 degrees and 25 degrees from pH 4 to 8.5. Corresponding measuremenportion of tRNA-Gly2-GGA/G molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNA-Gly2-GGA/G(nucleotide position 38). Asecondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNA-Gly2-suA36(HA), suggesting that the enzymes responsible for this modification recognize the anticodon sequencesof prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNA-Gly2-suA36(HA), by an alteration of the anticodon sequence of tRNA-Gly2-GGA/G is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions may be recognized by the glycyl-tRNA synthetase; the involvement of theanticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNA-Gly2-suA36(HA).     

Escherichia coli supH suppressor: temperature-sensitive missense suppression caused by an anticodon change in tRNASer2.

We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes. supH is a mutant form of serU which codes for tRNASer2. The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon. The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon. We propose that the supH suppressor causes the insertion of serine in response to this codon. The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species.     

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.     

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     

Suppression of a double missense mutation by a mutant tRNA(Phe) in Escherichia coli.

We report here the isolation of a mutant tRNAPhe that suppresses a double missense auxotrophic mutation in trpA of Escherichia coli, trpA218. The doubly mutant protein product differs from wild-type TrpA by the replacements of Phe22 by Leu and Gly211 by Ser. A partial revertant TrpA phenotype can be obtained from trpA218 by changing either Leu22 back to Phe or Ser211 back to Gly. Translational suppressors were previously obtained that act at codon 211, replacing the Ser211 in the TrpA218 protein, presumably with Gly. In the present study, we selected for trpA218 suppressors caused by mutation of a cloned tRNAPhe gene, pheV. DNA sequence analysis of the suppressor isolated reveals a singular structural alteration, changing the anticodon from 5'-GAA-3' to 5'-GAG-3'. Sequencing of trpA218 confirmed the likely identity of Leu22 as CUC. The new missense suppressor, designated pheV(SuCUC), is lethal to the cell when highly expressed, as from a high copy number plasmid. This may be due to efficient replacement of Leu by Phe at CUC (and, probably, CUU) codons throughout the genome. We anticipate that pheV(SuCUC) will prove, like other missense suppressors, to be extremely useful in studies on the specificity and accuracy of decoding.     

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.     

Genetic analysis of structure and function in phage T4 tRNASer

We have determined the nucleotide sequences of 55 spontaneous mutations that inactivate a suppressor gene of phage T4 tRNASer. Most of the mutations caused substitutions or deletions of single nucleotides at 18 different positions in the tRNA. Two of three mutations that allowed the synthesis of mature tRNA had nucleotide substitutions at the junction of the dihydrouridine and anticodon stems, suggesting that this region of tRNASer is important for aminoacylation. The third mutation that synthesized tRNA had a nucleotide deletion in the anticodon loop, which presumably affected the translational capacity of the tRNA. We also sequenced 58 spontaneous reversion mutations derived from strains with the inactive suppressor genes. Some of these regenerated the initial tRNA sequence, while other generated a second-site mutation in the tRNA. These second-site mutations restored helical base-pairings to the tRNA that had been eliminated by the initial mutations. The new base-pairings involved G.C and A.U, and the A.C wobble pair at certain positions in the tRNA. This finding establishes the existence of A.C wobble pair in tRNA helices.     

Primary structure of an unusual glycine tRNA UGA suppressor.

We have determined the nucleotide sequences of two UGA-suppressing glycine transfer RNAs. The suppressor tRNAs were previously shown to translate both UGA and UGG and to have arisen as a consequence of mutation in glyT, the gene for the GGA/G-reading glycine tRNA of Escherichia coli. In each mutant tRNA, the primary sequence change was the substitution of adenine for cytosine in the 3' position of the anticodon. In addition, a portion of mutant glyT tRNA molecules contained N6-(delta 2-isopentenyl)-2-thiomethyl adenine adjacent to the 3' end of the anticodon (nucleotide 37). The presence or absence of this hypermodification may be a determinant in some of the biological properties of the mutant tRNA.     

Trans-acting RNA inhibits tRNA suppressor activity in vivo

We constructed two aptamers, each of which contains a 7-nt-long loop complementary to the anticodon loop of a suppressor tRNA. One of these aptamers can form a stable bimolecular complex with the suppressor tRNA in vitro and protects the 7 nt in the suppressor's anticodon loop from RNase S1. An Escherichia coli strain, carrying an amber mutation in the lac Z gene, produces beta-galactosidase only if the suppressor is present; the aptamer's coexpression in the cell inhibits the activity of the suppressor tRNA. Moreover, in E. coli extract, the aptamer partially inhibits the read-through of the stop codon on the part of the suppressor tRNA. These results point to a novel strategy that need not be limited to the suppressor tRNA. By constructing appropriate inducible aptamers, it may well be possible to effectively control translation in vivo.     

Loss of positional specificity in the aminoacylation of Escherichia coli tRNAGly

The positional specificity in the aminoacylation of Escherichia coli tRNAGly by its cognate aminoacyl-tRNA synthetase has been studied using tRNAGlys terminating in 2'- or 3'-deoxyadenosine under conditions believed to alter tRNA conformation. Although E. coli tRNAGly terminating in 3'-deoxyadenosine has been reported not to be a good substrate for activation by the homologous glycyl-tRNA synthetase, by systematic variation of the conditions employed for aminoacylation it was possible to activate this tRNA to essentially the same extent as unmodified tRNAGly. Activation of tRNAGly terminating in 3'-deoxyadenosine was carried out optimally at 45 degrees C in an incubation mixture containing 0.3-0.4 M NaCl; 10% methanol, ethanol, and dimethyl sulfoxide were found to facilitate activation of the modified tRNA. Interestingly, the conditions employed to enhance activation of this modified tRNAGly had no effect on the activation of unmodified tRNAGly or tRNAGly terminating in 2'-deoxyadenosine. These experiments afford insight into the activation of tRNAGly by glycyl-tRNA synthetase and provide facile access to positionally defined, isomeric glycl-tRNAGlys.