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.     

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.     

The mutA mistranslator tRNA-induced mutator phenotype requires recA and recB genes, but not the derepression of lexA-regulated functions

The mapping of mutA and mutC mutator alleles to the glyV and glyW glycine tRNA genes, respectively, and the subsequent discovery that the mutA phenotype is abolished in a DeltarecA strain raise the possibility that asp --> gly misinsertion may induce a novel mutagenic pathway. The recA requirement suggests three possibilities: (i) the SOS mutagenesis pathway is activated in mutA cells; (ii) loss of recA function interferes with mutA-promoted asp --> gly misinsertion; or (iii) a hitherto unrecognized recA-dependent mutagenic pathway is activated by translational stress. By assaying the expression levels of a reporter plasmid bearing a umuC :lacZ fusion, we show that the SOS regulon is not in a derepressed state in mutA cells. Neither overexpression of the lexA gene through a multicopy plasmid nor replacement of the wild-type lexA allele with the lexA1[Ind-] allele interferes with the expression of the mutA phenotype. The mutA phenotype is unaffected in cells defective for dinB, as shown here, and is unaffected in cells defective for umuD and umuC genes, as shown previously. We show that mutA-promoted asp --> gly misinsertion occurs in recA- cells and, therefore, the requirement for recA is 'downstream' of mistranslation. Finally, we show that the mutA phenotype is abolished in cells deficient for recB, suggesting that cellular recombination functions may be required for the expression of the mutator phenotype. We propose that translational stress induces a previously unrecognized mutagenic pathway in Escherichia coli.     

Can tRNAs act as antisense RNA? The case of mutA and dnaQ

Many mutator genes have been characterized in E. coli, but the realization that mutA, the most recent mutator pathway described, encodes for a missense suppressor glycine tRNA caused a real surprise. The connection between expression of mutA and a 10 times increase in the spontaneous mutation rate is not readily explainable. The first attempt to describe the mechanism of action suggested a direct mistranslation of one subunit of polymerase III (PolIII) and the ideal candidate was the epsilon subunit carrying the 3'-->5' exonuclease activity. This subunit increases PolIII accuracy about 100 times. However, such direct mistranslation of epsilon was later ruled out when it became clear that all mutA cells express an error-prone form of PolIII. This result could not be reconciled with the very low level of mistranslation (1%) caused by mutA. But there is no need to invoke amino acid misincorporation in epsilon to destroy its activity. On the contrary, I suggest a new way to regulate epsilon amount, based on the reinterpretation of the mutA pathway through the new and puzzling observation that several tRNAs (including mutA which encodes for a glycine missense suppressor tRNA) are complementary to the 5' end of dnaQ mRNA. Accordingly, I propose that uncharged tRNAs can act as antisense RNAs, decreasing translation of dnaQ and possibly other genes. This could represent a new regulatory function for tRNAs and of course gives a direct and unrecognized link between starvation and mutation rate.     

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.     

Hypermutagenesis in mutA cells is mediated by mistranslational corruption of polymerase, and is accompanied by replication fork collapse

Elevated mistranslation induces a mutator response termed translational stress‐induced mutagenesis (TSM) that is mediated by an unidentified modification of DNA polymerase III. Here we address two questions: (i) does TSM result from direct polymerase corruption, or from an indirect pathway triggered by increased protein turnover? (ii) Why are homologous recombination functions required for the expression of TSM under certain conditions, but not others? We show that replication of bacteriophage T4 in cells expressing the mutA allele of the glyV tRNA gene (Asp→Gly mistranslation), leads to both increased mutagenesis, and to an altered mutational specificity, results that strongly support mistranslational corruption of DNA polymerase. We also show that expression of mutA, which confers a recA‐dependent mutator phenotype, leads to increased lambdoid prophage induction (selectable in vivo expression technology assay), suggesting that replication fork collapse occurs more frequently in mutA cells relative to control cells. No such increase in prophage induction is seen in cells expressing alaV^Glu tRNA (Glu→Ala mistranslation), in which the mutator phenotype is recA‐independent. We propose that replication fork collapse accompanies episodic hypermutagenic replication cycles in mutA cells, requiring homologous recombination functions for fork recovery, and therefore, for mutation recovery. These findings highlight hitherto under‐appreciated links among translation, replication and recombination, and suggest that translational fidelity, which is affected by genetic and environmental signals, is a key modulator of replication fidelity.     

Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA-dependent error-prone DNA replication activity

A base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp --> gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes. Here, we demonstrate that mutA cells express an error-prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single-stranded viral DNA bearing a model mutagenic lesion to the double-stranded replicative form. Amplification of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation fixation at 3, N4-ethenocytosine (varepsilonC) was approximately 3% when the DNA was replicated by normal cell extracts, approximately 48% when replicated by mutA cell extracts and approximately 3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results. Mutagenesis at undamaged DNA sites was significantly elevated by mutA cell-free extracts in the M13 lacZ(alpha) forward mutagenesis system. Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modification of DNA polymerase III or the activation of a previously unidentified DNA polymerase. These findings define the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication.     

Natural homolog of tRNA synthetase editing domain rescues conditional lethality caused by mistranslation

AlaXp is a widely distributed (from bacteria to humans) genome-encoded homolog of the editing domain of alanyl-tRNA synthetases. Editing repairs the confusion of serine and glycine for alanine through clearance of mischarged (with Ser or Gly) tRNA(Ala). Because genome-encoded fragments of editing domains of other synthetases are scarce, the AlaXp redundancy of the editing domain of alanyl-tRNA synthetase is thought to reflect an unusual sensitivity of cells to mistranslation at codons for Ala. Indeed, a small defect in the editing activity of alanyl-tRNA synthetase is causally linked to neurodegeneration in the mouse. Although limited earlier studies demonstrated that AlaXp deacylated mischarged tRNA(Ala) in vitro, the significance of this activity in vivo has not been clear. Here we describe a bacterial system specifically designed to investigate activity of AlaXp in vivo. Serine toxicity, experienced by a strain harboring an editing-defective alanyl-tRNA synthetase, was rescued by an AlaXp-encoding transgene. Rescue was dependent on amino acid residues in AlaXp that are needed for its in vitro catalytic activity. Thus, the editing activity per se of AlaXp was essential for suppressing mistranslation. The results support the idea that the unique widespread distribution of AlaXp arises from the singular difficulties, for translation, poised by alanine.     

Wobble modification defect in tRNA disturbs codon-anticodon interaction in a mitochondrial disease

We previously showed that in mitochondrial tRNA(Lys) with an A8344G mutation responsible for myoclonus epilepsy associated with ragged-red fibers (MERRF), a subgroup of mitochondrial encephalomyopathic diseases, the normally modified wobble base (a 2-thiouridine derivative) remains unmodified. Since wobble base modifications are essential for translational efficiency and accuracy, we used mitochondrial components to estimate the translational activity in vitro of purified tRNA(Lys) carrying the mutation and found no mistranslation of non-cognate codons by the mutant tRNA, but almost complete loss of translational activity for cognate codons. This defective translation was not explained by a decline in aminoacylation or lowered affinity toward elongation factor Tu. However, when direct interaction of the codon with the mutant tRNA(Lys) defective anticodon was examined by ribosomal binding analysis, the wild-type but not the mutant tRNA(Lys) bound to an mRNA- ribosome complex. We therefore concluded that the anticodon base modification defect, which is forced by the pathogenic point mutation, disturbs codon- anticodon pairing in the mutant tRNA(Lys), leading to a severe reduction in mitochondrial translation that eventually could result in the onset of MERRF.     

Examination of the Role of DNA Polymerase Proofreading in the Mutator Effect of Miscoding tRNAs

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, , is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutD alleles. In the present work, we extended the study of specific mutD alleles and constructed 16 altered mutD genes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes . We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the subunit playing a major role in the mutator effect of misreading tRNAs.     

Red queen dynamics of protein translation

We explore adaptive theories for the diversity of translational binding based on the genetic code viewed as a primitive mechanism of resistance. Modifying the set of codons bound by tRNA anticodon molecules or changing the specificity of binding, reduces the replication rate of translational parasites such as viruses. Increased translational efficiency of the parasite requires a high degree of specificity of host tRNAs for the parasite codons. This suggests that the genetic code might serve as the first line of defense against infection. We construct a red queen theory for translational diversity: a theory in which host-translational strategies- as defined by the degree of redundancy (a single anticodon binding many codons for a single amino acid) or degeneracy (many anticodons binding many codons for a single amino acid)-are constantly shifting through time to evade parasitism but where neither parasite nor host gain a systematic advantage.     

A mutation in a methionine tRNA gene suppresses the prp2-1 Ts mutation and causes a pre-mRNA splicing defect in Saccharomyces cerevisiae.

The PRP2 gene in Saccharomyces cerevisiae encodes an RNA-dependent ATPase that activates spliceosomes for the first transesterification reaction in pre-mRNA splicing. We have identified a mutation in the elongation methionine tRNA gene EMT1 as a dominant, allele-specific suppressor of the temperature-sensitive prp2-1 mutation. The EMT1-201 mutant suppressed prp2-1 by relieving the splicing block at high temperature. Furthermore, EMT1-201 single mutant cells displayed pre-mRNA splicing and cold-sensitive growth defects at 18 degrees. The mutation in EMT1-201 is located in the anticodon, changing CAT to CAG, which presumably allowed EMT1-201 suppressor tRNA to recognize CUG leucine codons instead of AUG methionine codons. Interestingly, the prp2-1 allele contains a point mutation that changes glycine to aspartate, indicating that EMT1-201 does not act by classical missense suppression. Extra copies of the tRNA(Leu)(UAG) gene rescued the cold sensitivity and in vitro splicing defect of EMT1-201. This study provides the first example in which a mutation in a tRNA gene confers a pre-mRNA processing (prp) phenotype.     

Evidence for a unique first position codon-anticodon mismatch in vivo

The Ser68(AGC) codon of the beta-lactamase gene was changed to the glycine codons GGA and GGC. With glycine at position 68, beta-lactamase is inactive because it does not have a nucleophilic side-chain to function in the reaction mechanism. The mutant SG68(GGA) allele had no detectable beta-lactamase activity; however, the mutant SG68(GGC) did produce a small amount of activity. Both mutant alleles produce comparable amounts of beta-lactamase protein in a maxi-cell system. To identify why these two "same-sense" beta-lactamase mutants differ phenotypically, we introduced the alleles into Escherichia coli strains with mutations that affect translational fidelity. The rpsD mutation, which decreases fidelity, significantly increased activity with the SG68(GGC) allele, while the rpsL mutation, which increases translational fidelity, had little effect on the beta-lactamase activity. The rpsD and rpsL alleles had no effect on the SG68(GGA) allele. From the allele specificity of the activity produced by the bla mutants, and from the differential effect of translational fidelity on the activity of the SG68(GGC) allele, we infer that tRNA(GCU)Ser, the AGU/C reading tRNA(Ser), mistranslates SG68(GGC) at a frequency of about 0.1%, and subsequently produces active beta-lactamase. This is the first observation of an A/G wobble with a wild-type tRNA at the first position of the codon-anticodon interaction.     

Specific incorporation of glycine into bacterial lipopolysaccharide. Novel function of specific transfer ribonucleic acids.

It has been found that the bacterial endotoxins (lipopolysaccharides, LPSs) contain some amino acids and glycine is the most abundant amino acid in the polysaccharide core preparations of LPSs of gram-negative bacteria. Until now nothing was known about the mechanism of amino acid incorporation into the lipopolysaccharide core. We found that one out of three glycyl-tRNAs(Gly) from Escherichia coli is the donor of amino acid and is the substrate for a putative aminoacyl-tRNA:LPS transferase. We have isolated, purified this tRNA and determined its nucleotide sequence to be major E.coli tRNA(3Gly). This tRNA(Gly) (anticodon GCC) conserved the tRNA structural features. The assay for determination of the specific incorporation of glycine into the lipopolysaccharide was also invented and described.     

Formation and persistence of polyglutamine aggregates in mistranslating cells

In neurodegenerative diseases, including pathologies with well-known causative alleles, genetic factors that modify severity or age of onset are not entirely understood. We recently documented the unexpected prevalence of transfer RNA (tRNA) mutants in the human population, including variants that cause amino acid mis-incorporation. We hypothesized that a mistranslating tRNA will exacerbate toxicity and modify the molecular pathology of Huntington''s disease-causing alleles. We characterized a tRNA^Pro mutant that mistranslates proline codons with alanine, and tRNA^Ser mutants, including a tRNA^Ser_AGA G35A variant with a phenylalanine anticodon (tRNA^Ser_AAA) found in ∼2% of the population. The tRNA^Pro mutant caused synthetic toxicity with a deleterious huntingtin poly-glutamine (polyQ) allele in neuronal cells. The tRNA^Ser_AAA variant showed synthetic toxicity with proteasome inhibition but did not enhance toxicity of the huntingtin allele. Cells mistranslating phenylalanine or proline codons with serine had significantly reduced rates of protein synthesis. Mistranslating cells were slow but effective in forming insoluble polyQ aggregates, defective in protein and aggregate degradation, and resistant to the neuroprotective integrated stress response inhibitor (ISRIB). Our findings identify mistranslating tRNA variants as genetic factors that slow protein aggregation kinetics, inhibit aggregate clearance, and increase drug resistance in cellular models of neurodegenerative disease.     

In vivo translational inaccuracy in Escherichia coli: missense reporting using extremely low activity mutants of Vibrio harveyi luciferase

A convenient, sensitive assay for measurement of in vivo missense translational errors is reported that uses luciferase activity generated by mistranslation of a gene encoding an inactive mutant alpha chain of the Vibrio harveyi enzyme. Mutations were introduced at alpha45 His, a position known to be highly intolerant of amino acids other than histidine. To normalize for any variations in expression level, the concentration of wild-type luciferase alphabeta dimer was determined by a novel assay using co-refolding of active/wild-type beta enzyme subunits with inactive alpha subunits in lysate with an excess of exogenously added active alpha subunits. Four His alpha45 missense mutants of luciferase encoded by leucine codons (CUC, CUU, CUG, and UUG) had histidine misincorporation rates of 2.0 x 10(-6), 1.3 x 10(-6), 9.0 x 10(-8), and 1.5 x 10(-8) respectively, a variation of over 133-fold among synonymous codons. Any substantial contribution of mutation was ruled out by a Luria-Delbrück fluctuation test. The two leucine codons with the highest rates, CUU and CUC, have a single central-mismatch to the histidyl-tRNAQUG anticodon. Aminoglycoside antibiotics known to enhance mistranslation increased the error rate of the CUC codon more than those of the CUU and CUG codons, consistent with the hypothesis that CUC codon mistranslation arises primarily from miscoding events such as the selection of noncognate histidyl-tRNAQUG at the central position of the codon.     

Complete DNA sequence of the mitochondrial genome of the ascidian Halocynthia roretzi (Chordata, Urochordata)

The complete nucleotide sequence of the 14,771-bp-long mitochondrial (mt) DNA of a urochordate (Chordata)-the ascidian Halocynthia roretzi-was determined. All the Halocynthia mt-genes were found to be located on a single strand, which is rich in T and G rather than in A and C. Like nematode and Mytilus edulis mtDNAs, that of Halocynthia encodes no ATP synthetase subunit 8 gene. However, it does encode an additional tRNA gene for glycine (anticodon TCT) that enables Halocynthia mitochondria to use AGA and AGG codons for glycine. The mtDNA carries an unusual tRNA(Met) gene with a TAT anticodon instead of the usual tRNA(Met)(CAT) gene. As in other metazoan mtDNAs, there is not any long noncoding region. The gene order of Halocynthia mtDNA is completely different from that of vertebrate mtDNAs except for tRNA(His)-tRNA(Ser)(GCU), suggesting that evolutionary change in the mt-gene structure is much accelerated in the urochordate line compared with that in vertebrates. The amino acid sequences of Halocynthia mt-proteins deduced from their gene sequences are quite different from those in other metazoans, indicating that the substitution rate in Halocynthia mt-protein genes is also accelerated.     

Analysis of the roles of tRNA structure, ribosomal protein L9, and the bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA

A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping). Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing.Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing.