C to U editing of the anticodon of imported mitochondrial tRNA(Trp) allows decoding of the UGA stop codon in Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNA(Trp)(CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNA(Trp) undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.     

C to U editing stimulates A to I editing in the anticodon loop of a cytoplasmic threonyl tRNA in Trypanosoma brucei

Editing of tRNAs is widespread in nature and either changes the decoding properties or restores the folding of a tRNA. Unlike the phylogenetically disperse adenosine (A) to inosine (I) editing, cytosine (C) to uridine (U) editing has only been previously described in organellar tRNAs. We have shown that cytoplasmic tRNA(Thr)(AGU) undergoes two distinct editing events in the anticodon loop: C to U and A to I. In vivo, every inosine-containing tRNA(Thr) is also C to U edited at position 32. In vitro, C to U editing stimulates conversion of A to I at the wobble base. Although the in vivo and in vitro requirements differ, in both cases, the C to U change plays a key role in A to I editing. Due to an unusual abundance of A34-containing tRNAs, our results also suggest that the unedited and edited tRNAs are functional, each dedicated to decoding a specific threonine codon. C to U editing of cytoplasmic tRNA expands the editing repertoire in eukaryotic cells, and when coupled to A to I changes, leads to an interrelation between editing sites.     

End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae

All mitochondrial tRNAs in Leishmania tarentolae are encoded in the nuclear genome and imported into the mitochondrion from the cytosol. One imported tRNA (tRNA(Trp)) is edited by a C to U modification at the first position of the anticodon. To determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5'- and 3'-end maturation of tRNA(Trp)(CCA), tRNA(Ile)(UAU), tRNA(Gln)(CUG), tRNA(Lys)(UUU), and tRNA(Val)(CAC). Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs. tRNA(Gln) was mainly cytosolic in localization; tRNA(Ile) and tRNA(Lys) were mainly mitochondrial; and tRNA(Trp) and tRNA(Val) were shared between the two compartments. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNA(Ile) underwent importation in vitro into isolated mitochondria more efficiently than 5'-extended precursor tRNA(Ile). 5'-Extended precursor tRNA(Trp) was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNA(Trp) was imported in vitro more efficiently than edited tRNA(Trp), suggesting the presence of importation determinants in the anticodon.     

Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons. The effect of modified bases adjacent to the anticodon triplet

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.     

Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.     

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.     

Taurine-containing uridine modifications in tRNA anticodons are required to decipher non-universal genetic codes in ascidian mitochondria

Variations in the genetic code are found frequently in mitochondrial decoding systems. Four non-universal genetic codes are employed in ascidian mitochondria: AUA for Met, UGA for Trp, and AGA/AGG(AGR) for Gly. To clarify the decoding mechanism for the non-universal genetic codes, we isolated and analyzed mitochondrial tRNAs for Trp, Met, and Gly from an ascidian, Halocynthia roretzi. Mass spectrometric analysis identified 5-taurinomethyluridine (τm(5)U) at the anticodon wobble positions of tRNA(Met)(AUR), tRNA(Trp)(UGR), and tRNA(Gly)(AGR), suggesting that τm(5)U plays a critical role in the accurate deciphering of all four non-universal codes by preventing the misreading of pyrimidine-ending near-cognate codons (NNY) in their respective family boxes. Acquisition of the wobble modification appears to be a prerequisite for the genetic code alteration.     

Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.     

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.     

The role of modifications in codon discrimination by tRNA(Lys)UUU

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.