Splicing of yeast tRNA precursors: a two-stage reaction.

Soluble extracts of S. cerevisiae splice tRNA precursors which contain intervening sequences. The reaction goes to completion and requires ATP for the production of mature sequence tRNA. In the absence of ATP, half-tRNA molecules accumulate. Similar half-tRNA molecules appear as kinetic intermediates and accumulate if splicing is inhibited with pure, mature tRNA. Half-tRNA molecules have been purified. These half-tRNAs are efficiently ligated in an ATP-dependent reaction that is inhibited by added mature tRNA. The product of ligation is the expected mature sequence tRNA. The excised intervening sequence has also been identified. These results suggest an enzymatic mechanism for splicing which involves two independent steps.     

Isolation and characterization of a guanine insertion enzyme, a specific tRNA transglycosylase, from Escherichia coli.

A guanine insertion enzyme (tRNA transglycosylase) was purified to a homogeneous state from Escherichia coli B by ammonium sulfate fractionation and DEAE-cellulose, DEAE-Sephadex A-50, phosphocellulose, and Sephadex G-200 column chromatographies. The molecular weight of the enzyme, which appeared to be a single polypeptide, was 4.6 X 10(4) by sodium dodecyl sulfate gel electrophoresis. The enzyme catalyzes exchange of guanine with guanine located in the first position of the anticodon of tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp, but unlike the enzymes isolated from rabbit reticulocytes and Ehrlich ascites tumor cells it does not catalyze the exchange of guanine with queuine (7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine) present in these tRNAs. The pH optimum of the reaction was 7.0, and the pH1 value was 4.6 to 4.8. The reaction required Mg2+ ion. 7-Methylguanine inhibited guanine insertion, but the other purine analogues tested were not inhibitory and could not replace guanine.20     

Splicing of yeast tRNA precursors: structure of the reaction intermediates.

The intermediates of the yeast tRNA splicing reaction have been characterized. The intervening sequence is excised as an unique linear molecule. It has 5'-hydroxyl and 3'-phosphate termini. Correspondingly, the half-tRNA molecules are shown to have a 3'-phosphate terminus on the 5' half and 5'-hydroxyl terminus on the 3' half. These isolated halves have been shown to be active in the ligation step of tRNA splicing. Removal of the 3'-phosphate from the 5' half eliminates the ability of the 5' half to participate in ligation.     

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.

tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.     

A new Heck reaction modification using ketone Mannich bases as enone precursors: parallel synthesis of anti-leishmanial chalcones

A new Heck-type reaction for the synthesis of chalcones has been established using Mannich bases as enone precursors. The novel reaction proceeds rapidly in air atmosphere under ligandless conditions and can be adapted for library synthesis in a parallel reactor station. Screening of the synthesized chalcones revealed N-{4-[(1E)-3-oxo-3-(3-pyridinyl)-1-propenyl]phenyl}benzamide (3f) to be a potent anti-leishmanial agent.     

Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase

In most organisms, the widely conserved 1-methyl-adenosine58 (m¹A₅₈) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m¹A₅₇ being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (_PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. _PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A₅₇ but not A₅₈, confirming that _PabTrmI methylates efficiently the first adenine of the A₅₇A₅₈A₅₉ sequence. _PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m¹A₅₈ formation triggers RNA release. A model of the protein–tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.     

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

Substitution of the queuine nucleobase precursor preQ₁ by an azide-containing derivative (azido-propyl-preQ₁) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNA^Asp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.     

The effect of specific structural modification on the biological activity of E. coli arginine tRNA.

Escherichia coli arginine tRNA1 has been modified at position s2C32 with iodoacetamide and a spin labelled derivative. The small effects on the charging ability of tRNA by the modifiications suggest that the synthetase does not bind to the tRNA in this region of the anticodon loop before the anticodon. A ternary complex of elongation factor Tu, GTP and the modified Arg-tRNA, can be formed allowing future studies of enzymatic binding to the ribosome. Using the triplet binding assay the native Arg-tRNA1 decodes all 4 codons beginning with CG. The modified Arg-tRNA1 has a restricted decoding but the decoding pattern is still unusual according to the Wobble Hypothesis.     

Agmatine is transported into liver mitochondria by a specific electrophoretic mechanism

Agmatine, a divalent diamine with two positive charges at physiological pH, is transported into the matrix of liver mitochondria by an energy-dependent mechanism the driving force of which is DeltaPsi (electrical membrane potential). Although this process showed strict electrophoretic behaviour, qualitatively similar to that of polyamines, agmatine is most probably transported by a specific uniporter. Shared transport with polyamines by means of their transporter is excluded, as divalent putrescine and cadaverine are ineffective in inhibiting agmatine uptake. Indeed, the use of the electroneutral transporter of basic amino acids can also be discarded as ornithine, arginine and lysine are completely ineffective at inducing the inhibition of agmatine uptake. The involvement of the monoamine transporter or the existence of a leak pathway are also unlikely. Flux-voltage analysis and the determination of activation enthalpy, which is dependent upon the valence of agmatine, are consistent with the hypothesis that the mitochondrial agmatine transporter is a channel or a single-binding centre-gated pore. The transport of agmatine was non-competitively inhibited by propargylamines, in particular clorgilyne, that are known to be inhibitors of MAO (monoamine oxidase). However, agmatine is normally transported in mitoplasts, thus excluding the involvement of MAO in this process. The I2 imidazoline receptor, which binds agmatine to the mitochondrial membrane, can also be excluded as a possible transporter since its inhibitor, idazoxan, was ineffective at inducing the inhibition of agmatine uptake. Scatchard analysis of membrane binding revealed two types of binding site, S1 and S2, both with mono-co-ordination, and exhibiting high-capacity and low-affinity binding for agmatine compared with polyamines. Agmatine transport in liver mitochondria may be of physiological importance as an indirect regulatory system of cytochrome c oxidase activity and as an inducer mechanism of mitochondrial-mediated apoptosis.     

Sequence specific purification of a particular tRNA by solid phase DNA probe.

A novel method for the purification of a specific tRNA using solid phase DNA probe is developed. With this method, the probe DNA immobilized on HPLC gel hybridized with target tRNA within a minute at room temperature. The hybridizing capacity of the solid phase probe was about 20 O.D. per gram dry gel when yeast phenylalanine tRNA was used. The specificity of this method was extremely high and the recovery rate was about 90%.     

Comparison of the tertiary structure of yeast tRNA(Asp) and tRNA(Phe) in solution. Chemical modification study of the bases

A comparative study of the solution structures of yeast tRNA(Asp) and tRNA(Phe) was undertaken with chemical reagents as structural probes. The reactivity of N-7 positions in guanine and adenine residues was assayed with dimethylsulphate and diethyl-pyrocarbonate, respectively, and that of the N-3 position in cytosine residues with dimethylsulphate. Experiments involved statistical modifications of end-labelled tRNAs, followed by splitting at modified positions. The resulting end-labelled oligonucleotides were resolved on polyacrylamide sequencing gels and analysed by autoradiography. Three different experimental conditions were used to follow the progressive denaturation of the two tRNAs. Experiments were done in parallel on tRNA(Asp) and tRNA(Phe) to enable comparison between the two solution structures and to correlate the results with the crystalline conformations of both molecules. Structural differences were detected for G4, G45, G71 and A21: G4 and A21 are reactive in tRNA(Asp) and protected in tRNA(Phe), while G45 and G71 are protected in tRNA(Asp) and reactive in tRNA(Phe). For the N-7 atom of A21, the different reactivity is correlated with the variable variable loop structures in the two tRNAs; in the case of G45 the results are explained by a different stacking of A9 between G45 and residue 46. For G4 and G71, the differential reactivities are linked to a different stacking in both tRNAs. This observation is of general significance for helical stems. If the previous results could be fully explained by the crystal structures, unexpected similarities in solution were found for N-3 alkylation of C56 in the T-loop, which according to crystallography should be reactive in tRNA(Asp). The apparent discrepancy is due to conformational differences between crystalline and solution tRNA(Asp) at the level of the D and T-loop contacts, linked to long-distance effects induced by the quasi-self-complementary anticodon GUC, which favour duplex formation within the crystal, contrarily to solution conditions where the tRNA is essentially in its free state.     

tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase. Assay development and characterization of the recombinant enzyme

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA(Tyr) in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-(14)C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg(2+) and Mn(2+) in millimolar concentrations. The steady-state kinetic parameters were determined to be K(m)(AdoMet) = 101.4 microm, K(m)(tRNA) = 1.5 microm, and k(cat) = 2.5 min(-1). A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K(m)(RNA) = 37.7 microm and k(cat) = 14.7 min(-1)), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.     

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)     

Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain.

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.     

The bases of the tRNA anticodon loop are independent by genetic criteria.

We employed two methods to study the translational role of interactions between anticodon loop nucleotides. Starting with a set of previously constructed weakly-suppressing anticodon loop mutants of Su7, we searched for second-site revertants that increase amber suppressor efficiency. Though hundreds of revertants were characterized, no second-site revertants were found in the anticodon loop. Second site reversion was detected in the D-stem, thereby demonstrating the efficacy of the search method. As a second method for detecting interactions, we used site-directed mutagenesis to construct multiple mutations in the anticodon loop. These multiple mutants are very weak suppressors and have translational activities that are equal to or lower than that predicted for the independent action of single mutations. We conclude that although the anticodon loop sequence of Su7 has an optimal structure for the translation of amber codons, we find no evidence that interactions between loop bases can enhance translational efficiency.     

tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition.

In eubacteria, the biosynthesis of queuine, a modified base found in the wobble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway. One of the key enzymes in this pathway, tRNA-guanine transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1. Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the second position of its seven base anticodon loop. Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited structural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E. coli tRNA(Tyr) minihelix. The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants. Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU(+1)). The other analogs were not active. This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem. In E. coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33. U33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. We find that mutation of this base confers on TGT the ability to recognize U34G35U36, and suggests that loop conformation affects recognition. The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate.     

Chorismic acid, a key metabolite in modification of tRNA.

Chorismic acid is the common precursor for the biosynthesis of the three aromatic amino acids as well as for four vitamins. Mutants of Escherichia coli defective in any of the genes involved in the synthesis of chorismic acid are also unable to synthesize uridine 5-oxyacetic acid (cmo5U) and its methyl ester (mcmo5U). Both modified nucleosides are normally present in the wobble position of some tRNA species. Mutants defective in any of the specific pathways leading to phenylalanine, tyrosine, tryptophan, folate, enterochelin, ubiquinone, and menaquinone have normal levels of cmo5U and mcmo5U in their tRNA. The presence of shikimic acid in the growth medium restores the ability of an aroD mutant to synthesize cmo5U, while O-succinylbenzoate, which is an early intermediate in the synthesis of menaquinone, does not. Thus, chorismic acid is a key metabolite in the synthesis of these two modified nucleosides in tRNA. The absence of chorismic acid blocks the formation of cmo5U and mcmo5U at the first step, which might be the formation of 5-hydroxyuridine. This results in an unmodified U in the wobble position of tRNA(1Val) and in most of the tRNAs normally containing cmo5U and mcmo5U. Since cmo5U and mcmo5U are synthesized under anaerobic conditions, the formation of these nucleosides does not require molecular oxygen. One of the carbon atoms of the side chain, --O--CH2--COOH, originates from the methyl group of methionine. The other carbon atom does not originate directly from the C-1 pool, from the carboxyl group methionine, or from bicarbonate. This metabolic link between intermediary metabolism and translation also exists for another member of the family Enterobacteriaceae, Salmonella typhimurium, as well as for the distantly related gram-positive organism Bacillus subtilis.