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PubMed1995

Structure of the P1 helix from group I self-splicing introns.

The upstream cleavage site of group I self-splicing introns is identified by an absolutely conserved U.G base-pair within a double helix. Mutant introns with a wobble C.A substitute are catalytically active, but all other combinations of nucleotides at these positions abolish splicing, suggesting that an unusual RNA structure generated by the wobble pair is recognized by the catalytic intron core. The solution structure of a 20-mer oligonucleotide containing a UUCG tetraloop hairpin and a U.G wobble pair within a double helix was determined by NMR spectroscopy without any assumptions on RNA conformation. Isotopically (15N/13C)-labelled RNA was used to collect an unusually large number of experimental constraints (703 in total, corresponding to approximately 35 constraints per nucleotide) leading to the determination of a structure with very high precision (overall root-mean-square-deviation (rmsd) between 20 converged structures 1.22 A, local rmsd 0.6 A for the tetraloop and 0.85 A for the stem). Analysis of the double helical structure at the conserved U.G wobble pair reveals local distortions from the regular A-form pattern, that may constitute the characteristic feature of U.G wobble pair recognized by the group I intron core and by amino acyl tRNA synthetases. Re-examination of the previously determined tetraloop structure reveals a novel U.G base-pair with a syn guanosine and hydrogen bonding contacts involving both base protons and a sugar 2'-OH. This explains the great stability of RNA UUCG loops when compared with DNA loops of identical sequence, and is one of the first NMR observations of RNA 2'-OH resonances.

Base Composition↗
PubMed1999

NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: the most conserved elements of structure are the most stable to unfolding.

The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded beta-barrel capped by a poorly conserved alpha-helix between strands beta3 and beta4. Two additional alpha-helices, unique to the LysN structure, flank the N terminus of the OB-fold. The stability of LysN to unfolding has been investigated with NMR native state hydrogen exchange measurements as a function of guanidinium hydrochloride concentration, and equilibrium unfolding transitions monitored by ellipticity at 222 nm and fluorescence at 350 nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with DeltaGu(0) approximately 6 kcal/mol and m approximately 3 kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of DeltaGu(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest DeltaGu(0) values from hydrogen exchange are for residues in the beta-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4 kcal/mol higher than those obtained from circular dichroism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with spectrophotometric parameters that reflect an unresolved super position of unfolding transitions of the alpha-helices and beta-strands. The largest DeltaGu(0) values obtained from hydrogen exchange for the subset of residues in the alpha-helices of the protein, agree with the DeltaGu(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three alpha-helices of LysN are on average 3 kcal/mol less stable than the beta-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first alpha-helix of the protein retains a cooperatively folded structure. Taken together with previous results on the OB-fold proteins SN and CspA, the present results for LysN suggest that the most conserved elements of structure in the OB-fold motif are the most resistant to denaturation. In all three proteins, stability to denaturation correlates with sequence hydrophobicity.

Anticodon↗
PubMed1977

Manganese(II) as a paramagnetic probe of the tertiary structure of transfer RNA.

The effect of manganese on both the low field (10--15 ppm) and the high field (o--3 ppm) NMR spectra of unfractionated tRNA and yeast tRNAPhe has been investigated. Trace amounts of Mn2+ cause selective broadening of resonances which are assigned to specific tertiary interactions. The order in which resonances broaden is the same as the order in which they are stabilized by the addition of magnesium, namely s4U8 - A14, U33 and A58 - T54. From this we conclude that three of the strong binding sites probably are the same for both Mn2+ and Mg2+, and that these sites are located close to the tertiary interactions which are stabilized by the strongly bound metals. The broadening data, taken in conjunction with published X-ray data on yeast tRNAPhe, permit us to suggest some plausible locations for the strong binding sites.

Binding Sites↗
PubMed1990

Sequence requirements of the hammerhead RNA self-cleavage reaction.

A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.

Base Sequence↗
PubMed2004

Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model.

A mechanistic understanding of ribosome function demands knowledge of the conformational changes that occur during protein synthesis. One current model proposes a conformational switch in Helix 27 (H27) of 16S rRNA involved in the decoding of mRNA. This model was based on the behavior of mutations in the 912 region of H27 of Escherichia coli 16S rRNA, which were predicted to stabilize the helix in either of two alternative conformations. This interpretation was supported by evidence from both genetics and structural biochemistry. However, recently published X-ray crystallographic structures of the Thermus thermophilus 30S subunit at different stages of tRNA selection have raised doubts regarding the validity of this model. We have therefore revisited the model genetically by constructing a H27 quadruple mutation (C912G, C910G, G885C, and G887C), which would create multiple mismatches in the proposed alternative conformation without perturbing the native H27 conformation seen in the crystal structures. Inconsistent with the H27 switch model, cells containing pure populations of quadruple mutant ribosomes grow at essentially wild-type rates. The mutants used to construct the H27 switch model all carried A2058G in 23S rRNA and C1192U in 16S rRNA as selectable markers. The quadruple mutant carrying these additional marker mutations is deleterious, and we conclude that they have a synergistic effect when combined with other mutations and are not phenotypically silent. Their presence confounded the interpretation of the original mutant phenotypes and, in light of the viability of the quadruple mutant, we conclude that the genetic evidence no longer supports the model.

Anti-Bacterial Agents↗
PubMed2002

The linkage between magnesium binding and RNA folding.

Understanding the linkage between Mg(2+) binding and RNA folding requires a proper theoretical model describing the energetics of Mg(2+) binding to the folded and unfolded states of RNA. Our current understanding of Mg(2+) binding to these different RNA states derives from empirical thermodynamic models that depend on a number of unjustified assumptions. We present a rigorous theoretical model describing the linkage between RNA folding and magnesium ion binding. In this model, based on the non-linear Poisson-Boltzmann (NLPB) equation, the stabilization of RNA by Mg(2+) arises from two distinct binding modes, diffuse binding and site binding. Diffusely bound Mg(2+) are described as an ensemble of hydrated ions that are attracted to the negative charge of the RNA. Site-bound Mg(2+) are partially desolvated ions that are attracted to electronegative pockets on the RNA surface. We explore two systems, yeast tRNA(Phe) and a 58-nucleotide rRNA fragment, with different Mg(2+) binding properties. The NLPB equation accurately describes both the stoichiometric and energetic linkage between Mg(2+) binding and RNA folding for both of these systems without requiring any fitted parameters in the calculation. Moreover, the NLPB model presents a well-defined physical description of how Mg(2+) binding helps fold an RNA. For both of the molecules studied here, the relevant unfolded state is a disordered intermediate state (I) that contains stable helical secondary structure without any tertiary contacts. Diffusely bound Mg(2+) interact with these secondary structure elements to stabilize the I state. The secondary structural elements of the I state fold into a compact, native tertiary structure (the N state). Diffuse binding plays a dominant role in stabilizing the N state for both RNAs studied. However, for the rRNA fragment, site-binding to a location with extraordinarily high electrostatic potential is also coupled to folding. Our results suggest that much experimental data measuring the linkage between Mg(2+) binding and RNA folding must be reinterpreted.

Escherichia coli↗
PubMed1981

[Phenylalanyl-tRNA synthetase from E. coli MRE-600. Effect of chemical modification of lysine residues on the enzyme interaction with substrates].

The effect of modification of Phe-RSase from E. coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated. It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-[32P]-pyrophosphate exchange reaction. The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction. tRNAphe strongly protects the enzyme against inactivation. ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect. L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP. The dissociation constant of the Phe-RSase[14C]-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction. There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested. Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex. Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction. It is assumed that Phe-RSase from E. coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center.

Adenosine Triphosphate↗
PubMed1979

Stability of the unique anticodon loop conformation of E.coli tRNAfMet.

Initiator tRNAs have an anticodon loop conformation distinct from that of elongation tRNAs as detected by susceptibility to S1 nuclease. We now find the anticodon loop conformation of E. coli tRNAfMet to be stable under different salt conditions as detected by using S1 nuclease as a structural probe. In contrast, a conformational change is observed in the T- and D- loop of this tRNA in the absence of added Mg2+. This change can be suppressed by spermine. Even under those conditions effecting a change in T- and D- loop conformation, the anticodon loop does not change. This suggests that the conformational shift is controlled by Mg2+ and restricted to the D- and T- loop region only without affecting the anticodon domain. The use of S1 nuclease as a conformational probe requires the use of kinetic studies to determine the initial cleavage sites. Thus, the use of a strong inhibitor which immediately stops the action of this nuclease is necessary. ATP is shown to be such an inhibitor.

Anticodon↗
PubMed1997

A biologically active 53 kDa fragment of overproduced alanyl-tRNA synthetase from Thermus thermophilus HB8 specifically interacts with tRNA Ala acceptor helix.

The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced. The gene comprises 2646 bp, corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli . The T. thermophilus AlaRS was overproduced in E.coli , purified and characterized. It has high thermal stability up to approximately 65 degrees C, with a temperature optimum of aminoacylation activity at approximately 60 degrees C, and will be valuable for crystallization. The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k cat/ K M values of 118 000/s/M for alanine and 114 000/s/M for ATP. By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also overproduced and purified. It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNAAla, as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, base pair. The reduced binding strength of such microhelices to AlaN470 enabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mode and intermolecular recognition.

Alanine-tRNA Ligase↗
PubMed1985

Anticodon-anticodon interactions in solution. Studies of the self-association of yeast or Escherichia coli tRNAAsp and of their interactions with Escherichia coli tRNAVal.

The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNAAsp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNAAsp (GUC)-Escherichia coli tRNAVal (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNAAsp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant (Kass = 10(4)M-1 at 0 degrees C) is characteristic of a rather weak complex, when compared with that between tRNAAsp and tRNAVal (Kass = 4 X 10(6) M-1 at 0 degrees C), the effect being essentially linked to differences in the rate constant for dissociation. tRNAAsp split in the anticodon by T1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNAGly (GCC), E. coli tRNAVal (GAC) or E. coli tRNAAla (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNAAsp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNAAsp but its complex with E. coli tRNAVal was found to be only 1.5 times less stable than that between yeast tRNAAsp and E. coli tRNAVal. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNAAsp (in the presence of 1.6 M-ammonium sulphate and 3mM-spermine) revealed an important stabilization of the yeast and E. coli tRNAAsp duplexes or of their complexes with E. coli tRNAVal. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.

Anticodon↗
PubMed1983

Low molecular weight aspartyl-tRNA synthetase from porcine thyroid. Purification, characterization, and heterogeneity.

The total low molecular weight aspartyl-tRNA synthetase activity of porcine thyroid is distributed among four distinct forms, all of which are identical in size, as determined by gel filtration. The predominant form was purified 25,000-fold to near homogeneity. A high concentration of glycerol (25%, v/v) was required throughout the procedure to maintain stability. The native enzyme was of the alpha 2-type with a Mr = 120,000 estimated by gel filtration. Its subunits were Mr = 53,000 as determined using polyacrylamide gel electrophoresis under denaturing conditions. The enzyme had an isoelectric point of pH 5.4 and pH optimum that varied from pH 7.3 to 8.8 depending on the type of buffer present. The variation in pH optimum was related to a salt effect. All salts tested were inhibitory, with the degree of inhibition dependent on the anion present. Inorganic pyrophosphate was a particularly powerful inhibitor; Km values for aspartate and tRNAAsp were significantly reduced in the presence of inorganic pyrophosphatase. Evidence is presented that the allotropism of the low molecular weight forms is not due to phosphorylation, proteolytic degradation, or stable enzyme-substrate complexes.

Amino Acyl-tRNA Synthetases↗
PubMed1986

Use of binding energy in catalysis analyzed by mutagenesis of the tyrosyl-tRNA synthetase.

The utilization of enzyme-substrate binding energy in catalysis has been investigated by experiments on mutant tyrosyl-tRNA synthetases that have been generated by site-directed mutagenesis. The mutants are poorer enzymes because they lack side chains that form hydrogen bonds with ATP and tyrosine during stages of the reaction. The hydrogen bonds are not directly involved in the chemical processes but are at some distance from the seat of reaction. The free energy profiles for the formation of enzyme-bound tyrosyl adenylate and the equilibria between the substrates and products were determined from a combination of pre-steady-state kinetics and equilibrium binding methods. By comparison of the profile of each mutant with wild-type enzyme, a picture is built up of how the course of reaction is affected by the influence of each side chain on the energies of the complexes of the enzyme with substrates, transition states, and intermediates (tyrosyl adenylate). As the activation reaction proceeds, the apparent binding energies of certain side chains with the tyrosine and nucleotide moieties increase, being weakest in the enzyme-substrate complex, stronger in the transition state, and strongest in the enzyme-intermediate complex. Most marked is the interaction of Cys-35 with the 3'-hydroxyl of the ribose. Removal of the side chain of Cys-35 leads to no change in the dissociation constant of ATP but causes a 10-fold lowering of the catalytic rate constant. It contributes no net apparent binding energy in the E X Tyr X ATP complex and stabilizes the transition state by 1.2 kcal/mol and the E X Tyr-AMP complex by 1.6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed2004

A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase.

The modified nucleoside 1-methyladenosine (m(1)A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m(1)A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m(1)A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m(1)I57). In yeast, the formation of m(1)A58 is catalysed by the essential tRNA (m(1)A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m(1)A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m(1)A MTases is discussed in the context of different hypotheses of the tree of life.

Amino Acid Sequence↗
PubMed1983

Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation.

Oligodeoxynucleotide-directed mutagenesis has been used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the adenosine 5'-triphosphate (ATP) binding site. Deliberate attempts were made to alter rather than destroy enzymic activity so that kinetic measurements may be made to identify the subtle roles of the enzyme-substrate interactions in catalysis. Cys-35, the -SH group of which is involved in binding the 3'-OH of the ribose ring of ATP, has been mutated to a serine residue [Winter, G., Fersht, A. R., Wilkinson, A. J., Zoller, M., & Smith, M. (1982) Nature (London) 299, 756-758] or glycine residue. The mutant enzymes are less active than the wild type, and the reduction in activity can be attributed to a decrease in the value of kcat and an increase in KM. Thus, the interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state. Relative to its absence in the Gly-35 mutant, the side chain of Cys-35 is calculated to stabilize the transition state for pyrophosphate exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.

Amino Acid Sequence↗
PubMed1992

Quantitative analysis of transcription and RNA levels of 15 barley chloroplast genes. Transcription rates and mRNA levels vary over 300-fold; predicted mRNA stabilities vary 30-fold.

Higher plant plastid genomes encode rRNAs, tRNAs, and protein subunits of the RNA polymerase, ribosomes, and the photosynthetic apparatus which vary over 1000-fold in abundance. Quantitative analysis of transcription and RNA levels was carried out on 15 plastid genes which are located in 14 different transcription units covering 50% of the barley plastid genome. Transcription of 16S rRNA, trnfM-trnG, and trnK was high relative to most other plastid genes. Transcription of trnfM-trnG was 5 times greater than trnK indicating that differences in tRNA levels in plastids could be due, in part, to differences in transcription. Among the protein coding genes, mRNA levels varied over 900-fold and transcription over 300-fold. The gene showing the lowest transcription rate and mRNA level, rpoB, is located in a gene cluster which encodes subunits of the plastid RNA polymerase (rpoB-rpoC1-rpoC2). RpoA, which encodes the alpha subunit of the RNA polymerase, was located in a gene cluster encoding ribosomal proteins (rpl23, rps19, rpl16) and infA. RNA from this gene cluster is 30-fold more abundant than rpoB mRNA, suggesting that expression of rpoA is regulated at the level of translation or protein stability. Polycistronic operons encoding subunits of the photosynthetic apparatus (psbB-psbH-petB-petD; psbK-psbI-psbD-psbC; atpB-atpE; psaA-psaB) had higher transcription rates and correspondingly higher mRNA levels than genes which encode ribosomal proteins or RNA polymerase subunits. RbcL and psbA, which are located in separate transcription units, exhibited the highest transcription rates and mRNA levels. Correspondence between transcription rate, mRNA level, and protein abundance indicates that transcription is a primary determinant of barley plastid gene expression. In addition, a 30-fold variation in predicted mRNA stability was observed which further increases the dynamic range of plastid mRNA abundance.

Base Sequence↗
PubMed1974

Protein synthesis in the cotyledons of Pisum sativum L. Protein factors involved in the binding of phenylalanyl-transfer ribonucleic acid to ribosomes.

1. Proteinaceous factors contained in a 0.5m-KCl extract of ribosomes from pea cotyledons form a ternary complex at 0 degrees C with [(14)C]phenylalanyl-tRNA and poly(U). The complex is measured by its quantitative retention on Millipore filters. 2. Complex-assembly is optimal at 5mm-Mg(2+) and is independent of GTP and ribosomes. 3. The addition of ribosomes is required to stabilize the complex at 34 degrees C. The complex binds to a puromycin-sensitive site on the ribosome. 4. Soluble factors from the 250000g supernatant of pea cotyledon form a Millipore-retainable complex dependent on GTP and ribosomes. 5. Complex-formation by soluble factors has a Mg(2+) optimum of 10-12mm and forms a puromycin-insensitive complex with ribosomes. 6. The function of the ribosomal protein factors and the supernatant fraction in initiation of protein synthesis is discussed.

Binding Sites↗
PubMed1999

Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR).

Post-transcriptional modifications are characteristic features of tRNAs and have been shown in a number of cases to influence both their structural and functional properties, including structure stabilization, amino-acylation and codon recognition. We have developed an approach which allows the investigation of the post-transcriptional modification patterns of human mitochondrial wild-type and mutant tRNAs at both the qualitative and the quantitative levels. Specific tRNA species are long-term labeled in vivo with [32P]orthophosphate, isolated in a highly selective way, enzymatically digested to mononucleotides and then subjected to two-dimensional thin layer chromatographic analysis. The wild-type tRNALysand the corresponding tRNALyscarrying the A8344G mutation associated with the MERRF (Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same modified nucleotides at the same molar concentrations. By contrast, a quantitatively different modification pattern was observed between the wild-type tRNALeu(UUR)and its counterpart carrying the A3243G mutation associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with Lactic Acidosis and Stroke-like episodes) syndrome, the latter exhibiting a 50% decrease in m2G content. Complementary sequencing of tRNALeu(UUR)has allowed the localization of this modification at position 10 within the D-stem of the tRNA. The decreased level of this modification may have important implications for understanding the molecular mechanism underlying the MELAS-associated mitochondrial dysfunction.

Autoradiography↗