TRNA genes transcribed from the plastid-like DNA of Plasmodium falciparum.
ABSTRACT: Besides their mitochondrial genome, malarial parasites contain a second organellar DNA. This 35 kb circular molecule has a number of features reminiscent of plastid DNAs. Sequence analysis shows that along with other genes the circle codes for 25 different tRNAs all of which are transcribed. Six of the tRNAs have some unusual features, and one has an intron, the only one found so far on the circle. Comparison of codon and anticodon usage indicates that the 25 tRNAs are sufficient to decode all the protein genes present on the circle. The maintenance of such a parsimonious but complete translation system is further evidence for the functionality of the circle.
Project description:The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons.
Project description:Some codons of the genetic code can be read not only by cognate, but also by near-cognate tRNAs. This flexibility is thought to be conferred mainly by a mismatch between the third base of the codon and the first of the anticodon (the so-called "wobble" position). However, this simplistic explanation underestimates the importance of nucleotide modifications in the decoding process. Using a system in which only near-cognate tRNAs can decode a specific codon, we investigated the role of six modifications of the anticodon, or adjacent nucleotides, of the tRNAs specific for Tyr, Gln, Lys, Trp, Cys, and Arg in Saccharomyces cerevisiae. Modifications almost systematically rendered these tRNAs able to act as near-cognate tRNAs at stop codons, even though they involve noncanonical base pairs, without markedly affecting their ability to decode cognate or near-cognate sense codons. These findings reveal an important effect of modifications to tRNA decoding with implications for understanding the flexibility of the genetic code.
Project description:Cellular health and growth requires protein synthesis to be both efficient to ensure sufficient production, and accurate to avoid producing defective or unstable proteins. The background of misreading error frequency by individual tRNAs is as low as 2 × 10(-6) per codon but is codon-specific with some error frequencies above 10(-3) per codon. Here we test the effect on error frequency of blocking post-transcriptional modifications of the anticodon loops of four tRNAs in Escherichia coli. We find two types of responses to removing modification. Blocking modification of tRNA(UUC)(Glu) and tRNA(QUC)(Asp) increases errors, suggesting that the modifications act at least in part to maintain accuracy. Blocking even identical modifications of tRNA(UUU)(Lys) and tRNA(QUA)(Tyr) has the opposite effect of decreasing errors. One explanation could be that the modifications play opposite roles in modulating misreading by the two classes of tRNAs. Given available evidence that modifications help preorder the anticodon to allow it to recognize the codons, however, the simpler explanation is that unmodified 'weak' tRNAs decode too inefficiently to compete against cognate tRNAs that normally decode target codons, which would reduce the frequency of misreading.
Project description:Some codons of the genetic code can be read not only by cognate, but also by near-cognate tRNAs. This flexibility is thought to be conferred mainly by a mismatch between the third base of the codon and the first of the anticodon (the so-called wobble position). However, this simplistic explanation underestimates the importance of nucleotide modifications in the decoding process. Using a system in which only near-cognate tRNAs can decode a specific codon, we investigated the role of six modifications of the anticodon, or adjacent nucleotides, of the tRNAs specific for Tyr, Gln, Lys, Trp, Cys and Arg in Saccharomyces cerevisiae. Modifications almost systematically rendered these tRNAs able to act as near-cognate tRNAs at stop codons, even though they involve non-canonical base-pairs, without markedly affecting their ability to decode cognate or near-cognate sense codons. These findings reveal an important effect of modifications to tRNA decoding with implications for understanding the flexibility of the genetic code. Overall design: Riboseq was done to study the readthrough after stop codons in WildType and Mutant.
Project description:Kinetoplastids encode a single nuclear tryptophanyl tRNA that contains a CCA anticodon able to decode the UGG codons used in cytoplasmic protein synthesis but cannot decode the mitochondrial UGA codons. Following mitochondrial import, this problem is circumvented in Trypanosoma brucei by specifically editing the tRNA(Trp) anticodon to UCA, which can now decode the predominant mitochondrial UGA tryptophan codons. This tRNA also undergoes an unusual thiolation at position 33 of the anticodon loop, the only known modification at U33 in any tRNA. In other organisms, tRNA thiolation is mediated by the cysteine desulfurase, Nfs1 (IscS). However, T. brucei encodes two Nfs homologues, one cytoplasmic and the other mitochondrial. We show by a combination of RNA interference and Northern and Western analyses that the mitochondria-targeted TbNfs, and not TbNfs-like protein, is essential for thiolation of both cytosolic and mitochondrial tRNAs. Given the exclusive mitochondrial localization of TbNfs, how it mediates thiolation in the cytoplasm remains unclear. Furthermore, thiolation specifically affects thiolated tRNA stability in the cytoplasm but more surprisingly acts as a negative determinant for the essential C to U editing in T. brucei. This provides a first line of evidence for mitochondrial C to U editing regulation in this system.
Project description:Five serine and three leucine isoaceptor tRNAs were purified from the asporogenic yeast Candida cylindracea, in which codon CUG is translated as serine instead of leucine, and their primary structures were determined. From the wobble hypothesis, it was assumed that one of the tRNA(Leu) species (Leu1), with the anticodon CmAA, corresponded to the UUG leucine codon, and that the remaining two leucine tRNAs (Leu2 and Leu3), with the same IAG anticodon sequence would decode the CUU, CUC and CUA codons as leucine, but not the CUG codon; this was clarified by an in vitro translation experiment with C.cylindracea using synthetic mRNAs containing the CUA or CUG codons. One of the serine tRNAs (Ser1) has already been demonstrated to have the anticodon CAG and to be responsible for translation of the codon CUG in C.cylindracea. Three of the other species of tRNA(Ser) (Ser2,3 and 4), with the anticodon sequences cm5UGA, IGA and CGA, can translate all four codons in the UCN codon box, while the remaining species (Ser5), with the anticodon GCU, corresponds to AGU and AGC serine codons. The gene sequences for these five serine and three leucine tRNAs were also determined, with the finding that only tRNA(Ser)CAG (Ser1) has an intron. At least five different types of tRNA(Ser)CAG genes exist in the genome of C.cylindracea. The nucleotide sequences of the flanking regions of these tRNA(Ser)CAG genes indicated that the tRNA(Ser)CAG gene has duplicated at least three times on the genome. The existence of multiple genes for tRNA(Ser)CAG on the genome may account for the observation that codon CUG is used very frequently in C.cylindracea. All of these tRNASerCAG genes contain the CCA sequence in their 3' termini, suggesting the possibility that during their multiplication process in the evolution of the C.cylindracea genome, the tRNA(Ser)CAG molecule was integrated into DNA via reverse transcription.
Project description:The position 34 of a tRNA is always modified for efficient recognition of codons and accurate integration of amino acids by the translation machinery. Here, we report genomics features of a deep-sea gut symbiotic Spiroplasma, which suggests that the organism does not require tRNA(34) anticodon modifications. In the genome, there is a novel set of tRNA genes composed of 32 species for recognition of the 20 amino acids. Among the anticodons of the tRNAs, we witnessed the presence of both U34- and C34-containing tRNAs required to decode NNR (A/G) 2:2 codons as countermeasure of probable loss of anticodon modification genes. In the tRNA fragments detected in the gut transcriptome, mismatches expected to be caused by some tRNA modifications were not shown in their alignments with the corresponding genes. However, the probable paucity of modified anticodons did not fundamentally change the codon usage pattern of the Spiroplasma. The tRNA gene profile that probably resulted from the paucity of tRNA(34) modifications was not observed in other symbionts and deep-sea bacteria, indicating that this phenomenon was an evolutionary dead-end. This study provides insights on co-evolution of translation machine and tRNA genes and steric constraints of codon-anticodon interactions in deep-sea extreme environment.
Project description:The malaria parasite Plasmodium falciparum carries an extrachromosomal 35 kb circular DNA molecule of unknown provenance. A striking feature of the circle is a palindromic sequence of genes for subunit rRNAs and several tRNAs, spanning ca. 10.5 kb. The palindrome has an intriguing resemblance to the inverted repeat of plastid genomes, and the sequence and putative secondary structure of the malarial large subunit (LSU) rRNA described in this report were used as the basis of a phylogenetic study. The malarial rRNA was found to be highly divergent in comparison with a selected group of chloroplast LSU rRNAs but was more closely related to them than to mitochondrial LSU rRNA genes.
Project description:The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA(Ala)GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA(Ala)GGC sequences. This suggests that the sequences of bacterial tRNA(Ala)GGC have evolved to avoid reading incorrect codons.
Project description:A lysate of purified mitochondria of the higher plant Oenothera processes in vitro synthesized tRNA precursors to the mature tRNA size. In vitro synthesized transcripts containing genuine plant mitochondrial tRNAs and analogous RNAs from mitochondrial loci with plastid derived tRNA sequences are accurately processed by an RNAase P-like activity to yield the mature 5'-terminus. A four nucleotide deletion in the anticodon stem-loop structure, however, prevents processing. The results show that in vitro transcripts containing tRNAs from sequence fragments of plastid origin integrated in plant mitochondrial genomes can be processed correctly in plant mitochondria, if tRNA sequences and structures are intact.