Reduced bacterial genomes and most genomes of cell organelles (chloroplasts and mitochondria) do not encode the full set of 32 tRNA species required to read all triplets of the genetic code according to the conventional wobble rules. Superwobbling, in which a single tRNA species that contains a uridine in the wobble position of the anticodon reads an entire four-fold degenerate codon box, has been suggested as a possible mechanism for how tRNA sets can be reduced. However, the general feasibility of superwobbling and its efficiency in the various codon boxes have remained unknown. Here we report a complete experimental assessment of the decoding rules in a typical prokaryotic genetic system, the plastid genome. By constructing a large set of transplastomic knock-out mutants for pairs of isoaccepting tRNA species, we show that superwobbling occurs in all codon boxes where it is theoretically possible. Phenotypic characterization of the transplastomic mutant plants revealed that the efficiency of superwobbling varies in a codon box-dependent manner, but—contrary to previous suggestions—it is independent of the number of hydrogen bonds engaged in codon-anticodon interaction. Finally, our data provide experimental evidence of the minimum tRNA set comprising 25 tRNA species, a number lower than previously suggested. Our results demonstrate that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA species utilizing standard base pairing or wobbling and by a second tRNA species employing superwobbling. This has important implications for the interpretation of the genetic code and will aid the construction of synthetic genomes with a minimum-size translational apparatus.

Reduced genomes of parasitic bacteria, chloroplasts, and mitochondria do not encode the full set of 32 tRNAs required to read all triplets of the genetic code according to Francis Crick's wobble rules. tRNAs with U in the wobble position of their anticodon might be able to make up for the deficit by pairing with any of the four bases at the third position of the codon via a mechanism called superwobbling. We have investigated the feasibility of superwobbling in the chloroplast genome of tobacco plants. We find that superwobbling occurs in all codon families where it is theoretically possible, demonstrating that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA utilizing standard base pairing or wobbling and by a second tRNA employing superwobbling. We also show that the efficiency of superwobbling is variable in different codon families. Finally, our data reveal that the minimum number of tRNAs required to sustain protein biosynthesis is 25.

Funding: This research was financed by the Max Planck Society and a grant from the Deutsche Forschungsgemeinschaft (FOR 804, BO 1482/15-2, to RB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We have previously shown that the unmodified uridine in the wobble position of the plastid tRNA-Gly(UCC) allows decoding of all four glycine codons (GGA, GGC, GGG and GGU; [10] ). Glycine codons are strong codons according to the “two-out-of-three” reading hypothesis. Here we have tested whether codon families of four mixed codons can be read by a single tRNA species. By systematically testing all tRNA species involved in reading four-fold degenerate codon boxes, we have established the complete set of decoding rules for the genetic system of the chloroplast. Moreover, we find that the efficiency of superwobbling varies in different codon boxes and is not directly correlated to the number of hydrogen bonds participating in the codon-anticodon interaction.

An alternative model of extended wobbling, referred to as the “two-out-of-three” reading hypothesis, was suggested by Lagerkvist [15] , [16] . This model defines “strong codons” as triplets with six hydrogen bonds formed by the first two bases of the codon in complementary base paring with the anticodon. In contrast, the first two bases of “mixed codons” have five and the first two bases of “weak codons” have four hydrogen bonds participating in base pairing [16] . The “two-out-of-three” reading hypothesis proposes that “strong codons” (with only G-C interactions between the first two bases of the anticodon and the first two bases of the codon) can be read by relying on base pairing with the first two bases of the anticodon, without a significant contribution of the interaction between the third codon position and the wobble position of the anticodon [15] . Due to their lower number of hydrogen bonds, codon boxes with “mixed codons” would be less likely to be readable by a single tRNA species [16] . In contrast, if the U in the wobble position contributes to the stability of the codon-anticodon interaction, as implied by the superwobble hypothesis, boxes with “mixed codons” may be readable with similar efficiency as “strong codons”.

32 tRNA species are needed to read all triplets of the genetic code according to the wobble rules proposed by Francis Crick [1] . However, reduced genomes, such as those of cell organelles (plastids and mitochondria) and some parasitic bacteria (e. g., mycoplasmas), contain fewer tRNA genes than this minimal set [2] . In mitochondria of plants and of some lineages of protozoa, at least some of the missing tRNA species are imported from the cytosol [3] , [4] and the possibility of tRNA import from the cytosol has also been suggested for plastids of parasitic plants [5] , [6] , [7] . However, tRNA import is unlikely to account for the seemingly incomplete tRNA sets in human mitochondria (encoding only 22 tRNA species), plastids (encoding 30 tRNA species) and parasitic bacteria [8] , [9] , [10] . In these systems, extended wobbling is believed to facilitate translation with a reduced set of tRNA species [9] . Extended wobbling refers to the ability of a single tRNA species to read all four triplets in a codon family. For example, uridine 5-oxyacetic acid at the wobble position enables a single tRNA to read all four triplets in a four-fold degenerate codon box [11] . Extended wobbling is also possible with an unmodified uridine in the wobble position of the anticodon and is also referred to as “four-way wobbling”, “hyperwobbling” or “superwobbling” [2] , [12] , [13] , [14] . Both theoretical considerations [1] and experimental data [10] support the idea that uridine in the wobble position of the anticodon can also engage in base-pairing interactions with U or C in the third codon position and, in this way promote reading of all four triplets in a codon family.

Results

Superwobbling in the ACN family of mixed codons To examine the possibility of superwobbling in mixed codons, we investigated the pair of threonine tRNAs encoded in the plastid (chloroplast) genome of higher plants. According to the conventional wobble rules [1], the tRNA-Thr(GGU) encoded by the plastid trnT-GGU gene should decode the two threonine triplets with a pyrimidine in third codon position (ACC and ACU), whereas the tRNA-Thr(UGU) encoded by the trnT-UGU gene should read the two threonine triplets with a purine in third codon position (ACA and ACG). The tRNA-Thr(UGU) has an unmodified uridine in the wobble position of the anticodon in all organelles and bacterial species with reduced tRNA sets (mycoplasmas), where its sequence was determined (http://trnadb.bioinf.uni-leipzig.de/). The assumption that the tRNA-Thr(UGU) can superwobble, but the tRNA-Thr(GGU) cannot (because, for steric reasons, the purines A and G in third codon position should not be capable of base pairing with the purine base guanine in the wobble position of the anticodon), would lead to the following testable predictions: (i) the trnT-GGU gene should be non-essential, (ii) the trnT-UGU should be essential, and (iii) the tRNA-Thr(UGU) alone should be sufficient to sustain plastid translation, at least to some extent. To test these predictions, we constructed knock-out alleles for both trnT genes in the plastid genome of the model plant tobacco (Nicotiana tabacum; Figure 1A–1D). The genes were disrupted by insertion of a selectable marker gene for chloroplast transformation (aadA) conferring spectinomycin resistance [17]. The knock-out alleles were then introduced into the plastid genome by particle gun-mediated transformation. Homologous recombination resulted in replacement of the wild-type allele with the knock-out allele. The resulting stably transformed (transplastomic) lines are subsequently referred to as ΔtrnT-UGU lines and ΔtrnT-GGU lines, respectively. Targeted disruption of the tRNA genes was confirmed by Southern blot analyses, which produced the expected restriction fragment length polymorphisms (Figure 1E, 1F). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Targeted inactivation of the two plastid trnT genes. (A) Physical map of the trnT-UGU containing region in the tobacco plastid genome (ptDNA; [44]). Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning or RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in ΔtrnT-UGU transplastomic plants. The selectable marker gene aadA (grey box) is inserted into the trnT-UGU gene in the same transcriptional orientation. (C) Physical map of the trnT-GGU containing region in the tobacco ptDNA. (D) Map of the transformed plastid genome in ΔtrnT-GGU plants. (E) RFLP analysis of ΔtrnT-UGU transplastomic lines. Independently generated transplastomic lines are designated by Arabic numerals following the tRNA gene name. All transplastomic lines remain heteroplasmic and show both the 1.9 kb wild type-specific hybridization band and the 3.1 kb band diagnostic of the transformed plastid genome. Wt: wild type. (F) RFLP analysis of ΔtrnT-GGU transplastomic plants. All lines are homoplasmic and show exclusively the 3.7 kb band diagnostic of the transgenic ptDNA. (G) Seed assays to confirm heteroplasmy of ΔtrnT-UGU plants and homoplasmy of ΔtrnT-GGU plants. Seeds were germinated in the absence or in the presence of spectinomycin. ΔtrnT-UGU plants produce mostly antibiotic-sensitive seedlings and a few antibiotic-resistant seedlings, as expected for a heteroplasmic situation. Moreover, many of the resistant seedlings are variegated indicating their composition of tissues possessing and tissues lacking the transgenic plastid genome. In contrast, the ΔtrnT-GGU lines produce homogeneous antibiotic-resistant progeny, confirming their homoplasmic status. (H) Analysis of tRNA-Thr(GGU) accumulation in the wild type, a heteroplasmic ΔtrnT-UGU line and a homoplasmic ΔtrnT-GGU line by northern blotting. Hybridization of electrophoretically separated RNA isolated from purified chloroplasts to a plastid trnT-GGU probe confirms complete lack of mature tRNA-Thr(GGU) in the ΔtrnT-GGU homoplasmic knock-out line, whereas its accumulation is unaltered in the heteroplasmic ΔtrnT-UGU line. Note accumulation of a ∼1.5 kb hybridizing RNA species in the ΔtrnT-GGU line, which corresponds to the tRNA-Thr(GGU) disrupted with the aadA cassette. To control for RNA loading, part of the ethidium bromide-stained gel (containing the two largest 23S rRNA hidden break products) prior to blotting is also shown. https://doi.org/10.1371/journal.pgen.1003076.g001 Due to the polyploidy of the plastid genome, the knock-out of an essential gene results in balancing selection for two antagonistic genome types: the wild-type genome (expressing the essential gene but not the antibiotic resistance) and the transformed genome (expression the antibiotic resistance but not the essential gene). Consequently, a stable mix of both genome types (heteroplasmy) is observed under antibiotic selection [18], [19], [20]. This stable heteroplasmy was clearly seen in all ΔtrnT-UGU lines (Figure 1E), consistent with the prediction that this gene should be essential. Growth in the absence of antibiotic selection releases the balancing selection in that it abrogates the selective pressure for maintenance of the transgenic plastid genome. This leads to predominant genome segregation towards the wild-type genome, which can be easily visualized by germinating seeds harvested from such plants on antibiotic-containing synthetic medium (where seedlings that lack the transgenic plastid genome bleach out; [21], [22]). This was observed in all ΔtrnT-UGU lines (Figure 1G), providing further evidence of the essentiality of the trnT-UGU gene. In contrast to trnT-UGU, the trnT-GGU gene turned out to be non-essential. Both DNA gel blot analyses and inheritance assays clearly demonstrated that homoplasmic knock-out plants had been obtained which lack residual wild-type copies of the plastid genome (Figure 1F, 1G). In addition, RNA gel blot analyses confirmed complete absence of tRNA-Thr(GGU) molecules from the ΔtrnT-GGU tobacco lines (Figure 1H), strongly suggesting that plastid translation can proceed in the absence of this tRNA species (and making it unlikely that this tRNA is imported from the cytosol). Thus, the tRNA-Thr(UGU) is necessary and sufficient to sustain translation, indicating that superwobbling in mixed codons is possible.

The single plastid alanine tRNA is essential The plastid alanine tRNA-Ala(UGC) may exemplify obligatory superwobbling in that there is only a single alanine tRNA species encoded in the chloroplast genome and it has a uridine in the wobble position of the anticodon. The U remains unmodified in mitochondria of the fungus Neurospora crassa as well as in the reduced bacterial genomes of the genus Mycoplasma (http://trnadb.bioinf.uni-leipzig.de/), but may carry an unknown modification in plastids [23]. Given the essentiality of plastid translation [19], [24] and the probable absence of tRNA import into plastids [8], [10], [22], this single alanine tRNA species is predicted to be encoded by an essential gene. To test this assumption, we constructed a knock-out allele for the trnA(UGC) gene and introduced it into the tobacco plastid genome (Figure S1). Molecular analysis of the generated ΔtrnA-UGC knock-out lines revealed stable heteroplasmy of the plastid genome under antibiotic selection (Figure S1D). Moreover, upon growth in soil, the heteroplastomic transplastomic plants displayed the characteristic leaf-loss phenotype (Figure S1F) caused by segregation into homoplasmy for the knock-out of an essential gene. This leads to cessation of cell division and ultimately to cell death, which in turn produces misshapen leaves that lack parts of their blade [20], [22], [24]. Finally, the ΔtrnA-UGC knock-out lines also showed a strong tendency to lose the transformed plastid genome in the absence of selective pressure, as evidenced by seed assays (Figure S1E). Taken together, these data suggest that the single plastid genome-encoded alanine tRNA is essential for translation and its loss cannot be complemented by tRNA import from the cytosol.

Wobbling makes the trnL-CAA gene dispensable Organellar genomes make maximum use of Crick's wobble rules [1] in that usually only a single tRNA species exists for each pair of codons (a pair being either the two triplets of a codon box with a purine in third position or the two triplets with a pyrimidine in third position). In the genomes of higher plant plastids, there is only a single exception: two distinct tRNA species exist for the two leucine codons UUA and UUG. These tRNAs are encoded by the trnL-UAA and trnL-CAA genes and the wobble positions of their anticodons are modified to 2′-O-methyluridine and 2′-O-methylcytidine, respectively [26]. Whether or not the base methylation in the wobble position enhances the specificity of decoding or perhaps even necessitates two distinct tRNA species for the reading of UUA and UUG triplets, is not known. To address these questions, we generated knock-out tobacco plants for the plastid trnL-UAA and trnL-CAA genes (Figure 4 and Figure S5). While homoplasmic knock-out lines were readily obtained for the trnL-CAA gene (Figure 4), the transplastomic ΔtrnL-UAA plants remained heteroplasmic and also fulfilled all other criteria of a mutant for an essential plastid gene (Figure S5). This indicates that the tRNA-Leu(UAA) can read both UUA and UUG triplets and suggests that 2′-O-methyluridine can wobble with G in the third codon position. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Targeted deletion of the plastid trnL-CAA gene. (A) Physical map of the region in the tobacco plastid genome containing the gene for trnL-CAA. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning and RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in ΔtrnL-CAA transplastomic plants. The aadA cassette replacing trnL-CAA is shown as grey box. (C) RFLP analysis of ΔtrnL-CAA plastid transformants. All lines are homoplasmic and show exclusively the 3.1-kb band diagnostic of the transplastome. Wt: wild type. (D) tRNA-Leu(CAA) accumulation in the wild type and ΔtrnL-CAA lines assessed by northern blotting. Hybridization to a plastid trnL-CAA probe confirms complete absence of the tRNA from homoplasmic knock-out lines. The ethidium bromide-stained agarose gel prior to blotting is also shown. (E) Confirmation of the homoplasmic state of the ΔtrnL-CAA lines by inheritance assays. Germination of seeds from transplastomic plants on spectinomycin-containing medium results in a homogeneous population of green antibiotic-resistant seedlings. (F) Wild-type seedlings are sensitive to spectinomycin and bleach out in the presence of the antibiotic. https://doi.org/10.1371/journal.pgen.1003076.g004 The four other leucine codons (in the CUN box) are most probably read by the third leucine tRNA species which is encoded by the plastid trnL-UAG gene. The tRNA-Leu(UAG) has an unmodified U in the wobble position (but an m7G modification in position 36 of the anticodon loop; [26], [23]). In the absence of a tRNA species with a GAG anticodon, the tRNA-Leu(UAG) should be capable of reading CUU and CUC triplets by superwobbling. As expected, transplastomic knock-out experiments confirmed essentiality of the trnL-UAG gene (Figure S6). Previous work has demonstrated that 7-methylguanosine at the wobble position allows base pairing with all four nucleotides, A, C, U and G [27]. In tRNA-Leu(UAG), the m7G modification is not in the wobble position, but in position 36, which base pairs with the first codon position. This raises the theoretical possibility that pairing of m7G with both C and U in first codon position could allow not only reading of CUN codons, but also of the two leucine codons in the UUN box, UUA and UUG. However, essentiality of the ΔtrnL-UAA (Figure S5) excludes this possibility and demonstrates that tRNA-Leu(UAG) cannot decode UUA and UUG triplets.