tRNA Gln Research Articles

Mitochondrial DNA polymorphisms and risk of Parkinson's disease in Spanish population

Mutations in mitochondrial DNA (mtDNA) have been implicated in the development of Parkinson's disease (PD). Mitochondrial function is necessary to supply the energy required for cell metabolism, and mutations in mitochondrial genes should have a deleterious effect in neuronal function. An association between several common mtDNA-polymorphisms and the risk of PD has been described. To test this association among Spanish patients, we genotyped 271 PD-patients and 230 healthy controls for 13 single-nucleotide polymorphisms (SNPs) through polymerase chain reaction (PCR) followed by digestion with a restriction enzyme. Alleles at eight of these SNPs define nine common European haplotypes, the mitochondrial haplogroups. In our population, no haplogroup showed significantly different frequencies between patients and controls. A significant association was found for the 4336T/C SNP (a polymorphism in the tRNA gln gene), with allele 4336C having a significantly increased frequency in PD-women compared to controls (OR = 4.45; 95%CI = 1.23–15.96; p = 0.011). We also sequenced five of the complex I genes (ND1 to ND5) in the patients who were 4336C, and no mutation in these genes was found. We also found a significantly reduced frequency of 10398G in patients ( p = 0.009; OR = 0.53), confirming a previously described protective effect for this allele in PD. In conclusion, we provided further evidence of the involvement of mitochondrial DNA variation in PD. In agreement with previous reports, we described a higher risk for PD among women with the mitochondrial 4336C allele in our population, and a protective effect for 10398G.

The complete set of tRNA species in Nanoarchaeum equitans

The archaeal parasite Nanoarchaeum equitans was found to generate five tRNA species via a unique process requiring the assembly of seperate 5′ and 3′ tRNA halves [Randau, L., Münch, R., Hohn, M.J., Jahn, D. and Söll, D. (2005) Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5′- and 3′-halves. Nature 433, 537–541]. Biochemical evidence was missing for one of the computationally-predicted, joined tRNAs designated as tRNA Trp. Our RT-PCR and sequencing results identify this tRNA as tRNA Lys (CUU) joined at the alternative position between bases 30 and 31. We show that the intron-containing tRNA Trp was misidentified in the initial Nanoarchaeum equitans genome annotation [E. Waters et al. (2003) The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc. Natl. Acad. Sci. USA 100, 12984–12988]. Along with a previously unidentified joined tRNA Gln (UUG), Nanoarchaeum equitans exhibits 44 tRNAs and is enabled to read all 61 sense codons. Features unique to this set of tRNA molecules are discussed.

Unusual usage of wobble modifications in mitochondrial tRNAs of the nematode Ascaris suum

To understand the decoding property of nematode mitochondrial tRNAs with unusual secondary structures, post-transcriptional modifications at wobble positions of Ascaris suum mitochondrial tRNAs corresponding to two-codon families ending with a purine were analyzed. 5-Carboxymethylaminomethyluridine (cmnm 5U) was identified at the wobble positions of tRNA Lys, tRNA Glu and tRNA Gln, while 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5s 2U) was present in tRNA UAA Leu and tRNA Trp . In most bacterial and mitochondrial tRNAs, the 2-thiouridine derivative is present in tRNAs for Lys, Glu and Gln. These is no report that cmnm 5s 2U is used in tRNA UAA Leu and tRNA Trp . The unusual usage of wobble modifications might assist decoding of nematode mitochondrial mRNAs.

Opening the black box: phylogenetics and morphological evolution of the Malagasy fossorial lizards of the subfamily “Scincinae”

The island of Madagascar harbors a highly endemic vertebrate fauna including a high diversity of lizards of the subfamily “Scincinae,” with about 57 species in eight genera. Since limb reduction seems to have been a common phenomenon during the evolution of Malagasy “scincines,” diagnosing evolutionary relationships based on morphology has been difficult. Phylogenetic analyses of multiple mitochondrial DNA sequences including the entire ND1, tRNA LEU, tRNA ILE, tRNA GLN genes, and fragments of the 12S and 16S rRNA and tRNA MET genes were conducted to test the monophyly of the largest genus Amphiglossus, and to evaluate the various formal and informal species groupings previously proposed for this species-rich group. A further objective was to determine the phylogenetic placements of the several greatly limb-reduced and limbless Malagasy “scincines” and ascertain whether any of these are derived from within the morphologically plesiomorphic Amphiglossus. As limb reduction in skinks is mostly associated with body elongation via an increase in the number of presacral vertebrae, we evaluate the pattern of evolution of the numbers of presacral vertebrae in the context of our phylogeny. We demonstrate that Amphiglossus as currently diagnosed is non-monophyletic, and the species fall into two major groups. One of these groups is a clade that contains the included species of the subgenus Amphiglossus ( Madascincus) among other species and is a member of a larger clade containing Paracontias and Pseudoacontias. In the second group, the nominate subgenus Amphiglossus ( Amphiglossus) forms several subclades within a larger clade that also contains Androngo crenni and Pygomeles braconnieri, and is sister to Voeltzkowia. All analyses provide strong support for the monophyly of Paracontias and Voeltzkowia. Based on the preferred phylogenetic hypothesis and weighted squared-change parsimony we show that the ancestor of the Malagasy clade was already elongated and had a moderately high number of presacral vertebrae (46–48), which is hypothesized to be the ancestral condition for the whole Malagasy “scincine” clade. We further demonstrate that both multiple increases and reductions of presacral vertebrae evolved in many clades of Malagasy “scincines” and that the use of presacral vertebrae as a major character to diagnose supraspecific units is dubious. Based on our results and published morphological evidence we consider Scelotes waterloti Angel, 1930 to be a junior synonym of Amphiglossus reticulatus (Kaudern, 1922).

The mitochondrial genome of the blowfly Chrysomya chloropyga (Diptera: Calliphoridae)

In view of the medical, sanitary and forensic importance of Chrysomya species, a knowledge of their nucleotide sequences would be useful for the molecular characterization of this genus, and would help in designing primers and in improving the molecular identification of Calliphoridae species. In this work, the mitochondrial genome of the blowfly Chrysomya chloropyga (Diptera: Calliphoridae) was completely sequenced. The entire mitochondrial DNA (mtDNA) molecule was 15,837 bp long and was sequenced using the shotgun approach. The overall nucleotide composition was heavily biased towards As and Ts, which accounted for 76.7% of the whole genome. The cox1 gene had a serine as the start codon, while incomplete termination codons mediated by tRNA signals were found for cox2, nd4 and nd5. The C. chloropyga genes were in the same order and orientation as the mitochondrial genome of other dipteran species, except for the occurrence of a 123 bp region that included a complete duplication of tRNA Ile and a partial duplication of tRNA Gln genes. C. chloropyga is the first species of Diptera with 23 tRNA genes instead of the usual 22 already described. A phylogenetic analysis showed a split of Brachycera into Calyptratae and Acalyptratae subdivisions. The complete sequence of C. chloropyga mtDNA described here will be a useful source of sequence information for general molecular and evolutionary studies in Diptera.

Analysis of murine leukemia virus replication complemented by yeast tRNA Phe reveals inherent preferences for the tRNA primer selected for reverse transcription

The replication of murine leukemia virus (MuLV) requires the capture of a cellular tRNA Pro as a primer for reverse transcription. To further study the specificity of primer selection, we have utilized a defective MuLV in which the primer-binding site (PBS) has been altered to be complementary to a nonmammalian tRNA, yeast tRNA Phe. Infectivity of the defective MuLV is dependent upon co-expression of yeast tRNA Phe in the cell. Defective MuLV genomes have been constructed in which the PBS was altered to be complementary to tRNA Phe that also encoded the cDNA for tRNA Phe. Transfection of these defective proviral genomes into cells resulted in the production of infectious MuLV as determined by a single-round assay. The amount of infectious virus produced using this complementation system, though, was approximately 6-fold lower than that produced following transfection of defective proviral genomes with a wild-type PBS complementary to tRNA Pro. The lower infectivity was not due to reduced expression of tRNA Phe in the transfected cells as compared to endogenous tRNA Pro or tRNA Lys,3. Serial passage of the MuLV genome with a PBS complementary to tRNA Phe that encoded tRNA Phe resulted in amplification of the virus. Using this rescue system, we have passaged the virus for four serial passages, after which time a revertant genome in which the PBS was altered to be complementary to tRNA Gln was detected that grew to high titers following subsequent serial passage. The results of these studies suggest that MuLV has preferences for the tRNA primer used in reverse transcription and are discussed with respect to the mechanism of primer selection.

Amino Acid Discrimination by a Class I Aminoacyl-tRNA Synthetase Specified by Negative Determinants

The 2.5 Å crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA Gln, an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft. In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain. The nucleophilic α-carboxylate moiety of glutamate is mispositioned with respect to both the ATP α-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction. Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH 2 group of the cognate glutamine substrate. At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule. These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested. Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation. The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases.

Dynamical analysis of tRNA Gln–GlnRS complex using normal mode calculation

We applied normal mode calculation in internal coordinates to a complex of glutamine transfer RNA (tRNA Gln) and glutaminyl-tRNA synthetase (GlnRS). Calculated deviations of atoms agreed well with those obtained from X-ray data. The differences of motions corresponding to low mode frequencies between the free state and the complex state were analyzed. For GlnRS, many motions in the free state were conserved in the complex state, while the dynamics of tRNA Gln was largely affected by the complex formation. Superimposed images of the conserved and non-conserved motions of tRNA Gln clearly indicated the restricted direction of motions in the complex.

Glutamine is incorporated at the nonsense codons UAG and UAA in a suppressor-free Escherichia coli strain

Readthrough of the nonsense codons UAG, UAA, and UGA is seen in Escherichia coli strains lacking tRNA suppressors. Earlier results indicate that UGA is miscoded by tRNA Trp. It has also been shown that tRNA Tyr and tRNA Gln are involved in UAG and UAA decoding in several eukaryotic viruses as well as in yeast. Here we have investigated which amino acid(s) is inserted in response to the nonsense codons UAG and UAA in E. coli. To do this, the stop codon in question was introduced into the staphylococcal protein A gene. Protein A binds to IgG, which facilitates purification of the readthrough product. We have shown that the stop codons UAG and UAA direct insertion of glutamine, indicating that tRNA Gln can read the two codons. We have also confirmed that tryptophan is inserted in response to UGA, suggesting that it is read by tRNA Trp.

Quality control of translation through the kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases.

It is known that each aminoacyl-tRNA synthetase (aaRS) specifically recognizes its cognate tRNAs to ensure the correct translation of the genetic information. However, we had previously demonstrated that mammalian mitochondrial seryl-tRNA synthetase (mt SerRS) can markedly misacylate mitochondrial (mt) tRNA(Gln). To investigate extensively misacylation reactions in mammalian mitochondrion, we purified overall twenty-two mt tRNAs from bovine liver, and determined their misacylation activities using mt SerRS. In addition to tRNA(Gln), tRNA(Ala) and tRNA(Asn) showed weak but significant serylation activities, which raises the possibility that each mammalian mt aaRS can misacylate several non-cognate mt tRNAs in varying degrees, but translational fidelity might be maintained by kinetic discrimination of tRNAs in the network of aaRSs.

Insertions in the anticodon loop of tRNA1Gln(sufG) and tRNA(Lys) promote quadruplet decoding of CAAA.

Base insertion mutations in the anticodons of two different Escherichia coli tRNAs have been isolated that allow suppression of a series of +1 frameshift mutations. Insertion of a U between positions 34 and 35 of tRNAGln1 or addition of a G between positions 36 and 37 of tRNA(Lys) expand the anticodons of both tRNAs similarly to 3'-GUUU(-5') and allow decoding of complementary 5'-CAAA(-3') quadruplets. Analysis of the suppressed mRNA sequences suggests that suppression occurs by pairing of the expanded anticodons to all four bases of the complementary, quadruplet codon. The tRNA Gln mutants are identical to the sufG class of frameshift suppressors isolated both in Salmonella enterica serovar Typhimurium and E. coli by Kohno and Roth and previously thought to affect tRNA(Lys).

Mitochondrial genetic code in cestodes

The flatworm mitochondrial genetic code, which has been used for all species of the Platyhelminthes, is mainly characterized by AUA codon for isoleucine, AAA codon for asparagine and UAA codon for tyrosine. In eight species of cestodes ( Echinococcus multilocularis, Echinococcus granulosus, Taenia solium, Taenia saginata, Taenia hydatigena, Taenia crassiceps, Hymenolepis nana and Mesocestoides corti), the cytochrome c oxidase subunit I (COI) genes were partially sequenced to verify this genetic code. Comparison of the COI-encoding nucleotide sequences with those of human, sea urchin, fruit fly, nematode and yeast indicated that the assignments of AUA and AAA codons are adequate for cestodes. In addition, the nucleotide sequences of ATPase subunit 6 (ATP6) gene and its flanking region were compared to examine initiation and stop codons. In the related species of T. solium and T. saginata, the deduced amino acid sequences of ATP6 were homogeneous; however, the conversion of initiation codon AUG into GUG was observed in T. saginata. We also found the similar conversion in T. crassiceps. The C-terminal sequences of putative ATP6 proteins were highly conserved among the eight species and the stop codon UAG was altered to UAA in all Taenia species. The features of the gene-junctional region between NADH dehydrogenase subunit 4 (ND4) and glutamine tRNA (tRNA Gln) genes also supported that UAA serves as a stop codon. Based on these results, we propose that the flatworm mitochondrial code should be modified for cestodes, particularly, in an initiating methionine codon (GUG) and a terminating codon (UAA).

Alternative designs for construction of the class II transfer RNA tertiary core

The structural requirements for assembly of functional class II transfer RNA core regions have been examined by sequence analysis and tested by reconstruction of alternative folds into the tertiary domain of Escherichia coli tRNA(2)Gln. At least four distinct designs have been identified that permit stable folding and efficient synthetase recognition, as assessed by thermal melting profiles and glutaminylation kinetics. Although most large variable-arm tRNAs found in nature possess an enlarged D-loop, lack of this feature can be compensated for by insertion of nucleotides either 3' to the variable loop or within the short acceptor/D-stem connector region. Rare pyrimidines at nt 9 in the core region can be accommodated in the class II framework, but only if specific nucleotides are present either in the D-loop or 3' to the variable arm. Glutaminyl-tRNA synthetase requires one or two unpaired uridines 3' to the variable arm to efficiently aminoacylate several of the class II frameworks. Because there are no specific enzyme contacts in the tRNAGln core region, these data suggest that tRNA discrimination by GlnRS depends in part on indirect readout of RNA sequence information.

Retracing the evolution of amino acid specificity in glutaminyl-tRNA synthetase

Molecular phylogenetic studies of glutaminyl-tRNA synthetase suggest that it has relatively recently evolved from the closely related enzyme glutamyl-tRNA synthetase. We have now attempted to retrace one of the key steps in this process by selecting glutaminyl-tRNA synthetase mutants displaying enhanced glutamic acid recognition. Mutagenesis of two residues proximal to the active site, Phe-90 and Tyr-240, was found to improve glutamic acid recognition 3–5-fold in vitro and resulted in the misacylation of tRNA Gln with glutamic acid. In vivo expression of the genes encoding these misacylating variants of glutaminyl-tRNA synthetase reduced cellular growth rates by 40%, probably as a result of an increase in translational error rates. These results provide the first biochemical evidence that glutaminyl-tRNA synthetase originated through duplication and consequent diversification of an ancestral glutamyl-tRNA synthetase-encoding gene.

Nucleotide sequences and functional characterization of two tobacco UAG suppressor tRNA(Gln) isoacceptors and their genes.

We isolated and sequenced the two major tRNA(Gln) isoacceptors with CUG and UmUG anticodons from the cytoplasm of Nicotiana rustica. These are the first tRNAs(Gln) of nuclear origin characterized in plants. The tRNA(Gln) sequences were used to design probes for the isolation of the corresponding genes from a nuclear DNA library of N. rustica. The two cloned Nicotiana tRNA(Gln) genes, coding for either of the two isoacceptors, are efficiently transcribed in HeLa cell nuclear extract. In vitro translation in the presence of purified Nicotiana tRNAs(Gln) was carried out in a wheat germ extract partially depleted of endogenous tRNAs. Cytoplasmic (cyt) tRNA(Gln)CUG and to a lesser extent cyt tRNA(Gln)UmUG stimulated readthrough over the UAG stop codon present in the tobacco mosaic virus-specific context. The two tRNA(Gln) isoacceptors are the second class of natural UAG suppressors identified in plants, in addition to cyt tRNA(Tyr)GpsiA which has previously been characterized as the first natural UAG suppressor.

MtDNA Polymorphisms in Japanese Sporadic Alzheimer’s Disease

We screened 92 Japanese patients with sporadic AD (clinically diagnosed: 72 cases; autopsy-confirmed: 20 cases) and 59 age-matched controls for mitochondrial polymorphisms previously reported to be associated with increased risk in Caucasian AD. The polymorphisms in tRNA Gln (nt. 4336), 16S rRNA (nt. 3196), and ND1 (nt. 3397) were not found in either in Japanese AD or age-matched controls. The frequencies of these polymorphisms in Japanese seems to be very rare if not absent, indicating that these three mutations are not likely to be genetic risk factors of Japanese AD. In the analysis of polymorphisms of 12S rRNA, however, we identified two novel mutations, an insertion of three cytosines and an A to G transition at nt. 856, which have not been described before. The insertion of three cytosines was observed in one of the 90 AD cases, but not in 59 normal controls. The A to G transition at nt. 856 was found in 2 of the 90 AD cases, but not in 59 normal controls. These results raise the possibility that the mutations in the 12S rRNA are genetic risk factors for AD in Japanese population.

Molecular cloning and sequencing of a nuclear gene encoding tRNA Gln (UUG) from Arabidopsis thaliana

A gene encoding cytoplasmic tRNA Gln (UUG) was isolated from an Arabidopsis DNA library. The coding sequence of the gene revealed 85% sequence identity with that of its animal counterparts. This is the first report of a nuclear tRNA Gln gene from higher plants.

Functional Connectivity Between tRNA Binding Domains in Glutaminyl-tRNA Synthetase

The structure of Escherichia coliglutaminyl-tRNA synthetase (GlnRS) in complex with tRNA Glnand ATP has identified a number a sequence-specific protein–tRNA interactions. The contribution to glutamine identity has previously been determined for the nucleotides in tRNA Gln. Here, we report the mutational analysis of residues in all three tRNA recognition domains of GlnRS, thus completing a survey of the major sequence-specific contacts between GlnRS and tRNA Gln. Specifically, we analyzed the GlnRS determinants involved in recognition of the anticodon which is essential for glutamine identity and in the communication of anticodon recognition to the acceptor binding domain in GlnRS. A combined in vivoand in vitroapproach has demonstrated that Arg341, which makes a single sequence-specific hydrogen bond with U35 in the anticodon of tRNA Gln, is involved in initial RNA recognition and is an important positive determinant for this base in both cognate and non-cognate tRNA contexts. However, Arg341, as well as Arg402, which interacts with G36 in the anticodon, are negative determinants for non-cognate nucleotides at their respective positions. Analysis of acceptor-anticodon binding double mutants and of a mutation of Glu323 in the loop-strand-helix connectivity subdomain in GlnRS has further implicated this domain in the functional communication of anticodon recognition. The better than expected activity (anticooperativity) of these double mutants has led us to propose an "anticodon-independent" mechanism, in which the removal of certain synthetase interactions with the anticodon eliminates structural constraints, thus allowing the relaxed specificity mutants in the acceptor binding domain to make more productive interactions.

A three-dimensional structure model of the complex of glutamyl-tRNA synthetase and its cognate tRNA

A docking model of glutamyl-tRNA synthetase (GluRS) and tRNA Glu was constructed, on the basis of the distinguished similarity between the X-ray crystallographic three-dimensional structures of the N-terminal halves of the Thermus thermophilus GluRS in the free state and the Escherichia coli glutaminyl-tRNA synthetase in a complex with tRNA Gln. The modeled structure is energetically favorable and is also well consistent with the results of site-directed mutagenesis studies. The model indicates that the GluRS-specific insertions 2 and 3 fit and bind to the acceptor stem and the D arm, respectively, of the cognate tRNA without affecting other contacts. In particular, insertion 3 strongly interacts with the two D-stem base pairs that are essential for the tRNA·GluRS recognition.

No association found between Alzheimer's disease and a mitochondrial tRNA glutamine gene variant

We have screened a large sample of patients with sporadic late-onset dementia of the Alzheimer type (DAT) and age-matched controls for a mitochondrial tRNA Gln variant previously reported to be associated with increased risk of developing Alzheimer's disease (AD). The frequency of an Ava II site gain was determined by restriction analysis of a PCR-amplified mitochondrial DNA product. One of 155 DAT cases and four of 105 age-matched controls carried the variant. Both the affected and control frequencies are statistically different from those previously reported. The mitochondrial lineage of those individuals harboring the variant was determined by sequencing a short region of the hypervariable mitochondrial D-loop. The affected individual and three of the four controls carrying the Ava II variant belong to the same mitochondrial lineage previously reported to be associated with AD.