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At least 19 recordsLinked to original sources
PubMed2000

Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases.

The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed2002

Role of editing in plant mitochondrial transfer RNAs.

Editing in plant mitochondria consists in C to U changes and mainly affects messenger RNAs, thus providing the correct genetic information for the biosynthesis of mitochondrial (mt) proteins. But editing can also affect some of the plant mt tRNAs encoded by the mt genome. In dicots, a C to U editing event corrects a C:A mismatch into a U:A base pair in the acceptor stem of mt tRNA(Phe) (GAA). In larch mitochondria, three C to U editing events restore U:A base pairs in the acceptor stem, D stem and anticodon stem, respectively, of mt tRNA(His) (GUG). For both these mt RNA(Phe) and tRNA(His), editing of the precursors is a prerequisite for their processing into mature tRNAs. In potato mt tRNA(Cys) (GCA), editing converts a C28:U42 mismatch in the anticodon stem into a U28:U42 non-canonical base pair, and reverse transcriptase minisequencing has shown that the mature mt tRNA(Cys) is fully edited. In the bryophyte Marchantia polymorpha this U residue is encoded in the mt genome and evolutionary studies suggest that restoration of a U28 residue is necessary when it is not encoded in the gene. However, in vitro studies have shown that neither processing of the precursor, nor aminoacylation of tRNA(Cys), requires C to U editing at this position. But sequencing of the purified mt tRNA(Cys) has shown that Psi is present at position 28, indicating that C to U editing is a prerequisite for the subsequent isomerization of U into Psi at position 28.

Cytidine↗
PubMed1990

Structure and expression of tomato mitochondrial genes coding for tRNA(Cys) (GCA), tRNA(Asn) (GUU) and tRNA(Tyr) (GUA): a native tRNA(Cys) gene is present in dicot plants but absent in monocot plants.

The nucleotide sequences of tRNA(Asn) (GUU) and tRNA(Tyr) (GUA) genes from tomato mitochondria and their flanking regions have been determined. The tomato mitochondrial tRNA(Asn) gene is located 2.1 kb downstream from the tRNA(Cys) gene reported previously (Izuchi and Sugita 1989) and shows a nearly complete identity with the corresponding chloroplast gene. The tRNA(Tyr) gene, which shows only 73% homology with the corresponding chloroplast gene, has to be considered a "native" mitochondrial tRNA gene and is 535 bp from the "chloroplast-like" tRNA(Asn) gene on the same strand. Northern hybridization analysis revealed that the three tRNA genes are transcribed in tomato mitochondria. Southern hybridization analysis of tomato, sugar beet. rice and wheat mitochondrial DNAs, with oligonucleotide probes for mitochondrial or chloroplast tRNA genes, demonstrated that the mitochondrial tRNA(Cys) gene found in tomato is present in dicot plants but not in monocots. On the other hand, a chloroplast-like tRNA(Cys) gene exists in monocot plants.

Base Sequence↗
PubMed1997

A strategy of tRNA recognition that includes determinants of RNA structure.

Recognition of tRNAs by aminoacyl tRNA synthetases establishes the connection between amino acids and anticodon triplets of the genetic code. Although anticodons and nucleotides adjacent to the amino acid attachment site are generally important, the tertiary structural framework of tRNAs has recently been implicated to have a role in tRNA recognition. A G15:G48 tertiary hydrogen base pair of E. coli tRNA(Cys) is important for recognition of the tRNA by cysteine tRNA synthetase. This base pair is proposed to consist of N2:N3, rather than N1:O6, hydrogen bonds. The reproduction of the hydrogen pairing scheme of tRNA(Gly). This reproduction required an A13:A22 mismatch in the dihyrouridine stem. To determine if A13:A22 is a determinant of the structural features of G15:G48, we investigated the A15:U48 and A15:A48 variants of tRNA(Gly) which harbored specific substitutions of A13:A22. We show here that introduction of A13:A22 to both tRNA frameworks confers structural features similar to those of G15:G48 in E. coli tRNA(Cys). These structural features are accompanied by efficient recognition of both tRNAs by cysteine tRNA synthetase. Substitution of A13:A22 with U13:A22 alters the structural features at 15:48 and impairs tRNA recognition. The dependence on A13:22 for tRNA recognition has a distinct similarity to that of E. coli tRNA(Cys) and to that of the G15:G48 variant of tRNA(Gly). The results have implications for the design and manipulation of RNA structural elements as the basis for tRNA recognition.

Acetylation↗
PubMed1996

RNA editing in the acceptor stem of squid mitochondrial tRNA(Tyr).

In squid (Loligo bleekeri) mitochondria, the two 3'-terminal nucleotides (G72-G73) of the tRNA(Tyr) gene overlap with the two 5'-terminal nucleotides (G1-G2) of the downstream tRNA(Cys) gene. To elucidate the processing mechanism(s) of the tRNA molecules derived from this region, tRNAs were analyzed by sequencing cDNAs synthesized from circularized tRNAs. Nucleotides G1-G2 in tRNA(Cys) appeared to be without post-transcriptional conversion, whereas CCA was post-transcriptionally added to the 3'-terminus. In contrast, in the majority of tRNAs(Tyr), G72-G73 were found to be converted to A72-A73, accompanied by the CCA addition. These results indicate that a precursor of tRNA(Tyr) is processed at U71 and two adenosines are attached prior to the CCA addition. Thus, we suggest that 5' processing of the precursor tRNA dominates 3' processing and maturation of the tRNA is mediated by a polyadenylylation enzyme in the mitochondria, a scenario which is consistent with the editing process proposed in land snail mitochondria. We also obtained intermediates, such as a premature tRNA lacking CCA that terminated at U71 and one with a single adenosine attached at position 72, which support the suggested maturation process. However, although we failed to detect a tRNA(Cys) lacking G1-G2 at the 5'-terminus, we obtained cDNAs for tRNA(Tyr) with G72-G73 and the CCA terminus. This inconsistent result suggests the co-existence of another process(es) in the maturation of these tRNA molecules in squid mitochondria.

Animals↗
PubMed1994

RNA editing of tRNA(Phe) and tRNA(Cys) in mitochondria of Oenothera berteriana is initiated in precursor molecules.

We have analyzed the role of RNA editing in the correction of mismatched base pairs in tRNA secondary structures in mitochondria of the flowering plant Oenothera berteriana. Comparison of genomic and cDNA sequences from unprocessed primary transcripts of the newly characterized genes for tRNA(Cys), tRNA(Asn) and tRNA(Ile) and the previously described gene for tRNA(Phe) revealed single nucleotide discrepancies in the tRNA(Cys) and tRNA(Phe) sequences. While the change in the anticodon stem of tRNA(Cys) alters a C-T to a T-T mismatch, the nucleotide transition in the tRNA(Phe) restores a conventional T-A Watson-Crick base pair, replacing a C-A mismatch in the acceptor stem. Since both nucleotide alterations are conversions from genomic cytidines to thymidines in the cDNA (uridines in the tRNAs), they are attributed to RNA editing, which is observed in nearly all mRNAs from plant mitochondria.

Base Sequence↗
PubMed1999

The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA.

BACKGROUND: . The translation elongation factor EF-Tu in its GTP-bound state forms a ternary complex with any aminoacylated tRNA (aa-tRNA), except initiator tRNA and selenocysteinyl-tRNA. This complex delivers aa-tRNA to the ribosomal A site during the elongation cycle of translation. The crystal structure of the yeast Phe-tRNAPhe ternary complex with Thermus aquaticus EF-Tu-GDPNP (Phe-TC) has previously been determined as one representative of this general yet highly discriminating complex formation. RESULTS: The ternary complex of Escherichia coli Cys-tRNACys and T. aquaticus EF-Tu-GDPNP (Cys-TC) has been solved and refined at 2.6 degrees resolution. Conserved and variable features of the aa-tRNA recognition and binding by EF-Tu-GTP have been revealed by comparison with the Phe-TC structure. New tertiary interactions are observed in the tRNACys structure. A 'kissing complex' is observed in the very close crystal packing arrangement. CONCLUSIONS: The recognition of Cys-tRNACys by EF-Tu-GDPNP is restricted to the aa-tRNA motif previously identified in Phe-TC and consists of the aminoacylated 3' end, the phosphorylated 5' end and one side of the acceptor stem and T stem. The aminoacyl bond is recognized somewhat differently, yet by the same primary motif in EF-Tu, which suggests that EF-Tu adapts to subtle variations in this moiety among all aa-tRNAs. New tertiary interactions revealed by the Cys-tRNACys structure, such as a protonated C16:C59 pyrimidine pair, a G15:G48 'Levitt pair' and an s4U8:A14:A46 base triple add to the generic understanding of tRNA structure from sequence. The structure of the 'kissing complex' shows a quasicontinuous helix with a distinct shape determined by the number of base pairs.

Amino Acid Sequence↗
PubMed2002

Prevention of mis-aminoacylation of a dual-specificity aminoacyl-tRNA synthetase.

Accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetase is essential for the fidelity of protein synthesis. For Methanococcus jannaschii tRNA(Pro), accuracy is difficult because the cognate prolyl-tRNA synthetase also recognizes and aminoacylates tRNA(Cys) with cysteine. We show here that the unmodified transcript of M. jannaschii tRNA(Pro) is indeed mis-acylated with cysteine. However, the origin of mis-charging is not at the anticodon or acceptor stem, the two hotspots for tRNA(Pro) and tRNA(Cys) identity determinants. Instead, replacement of the D loop in the tRNA core with that of tRNA(Cys) suppresses mis-charging with cysteine without compromising the activity of aminoacylation with proline. The reduced level of cysteine activity of the chimera is not due an editing response of the synthetase and is consistent with a relaxed sensitivity of the tRNA to the analog thiaproline in aminoacylation with cysteine. We suggest that mis-acylation is not due to the presence of cysteine determinants, but to a mis-placed 3' end into the cysteine catalytic site that activates and transfers cysteine to the tRNA. Prevention of mis-placement by alteration of the core structure or by nucleotide modifications in the tRNA illustrates a novel strategy of the dual-specificity synthetase.

Acylation↗
PubMed1992

RNA editing in trans-splicing intron sequences of nad2 mRNAs in Oenothera mitochondria.

The complete open reading frame of subunit 2 of the NADH dehydrogenase in Oenothera mitochondria is split into five exons. The first two and the last three exons are encoded in distant genomic locations and are transcribed separately. Three tRNA genes coding for tRNA(Cys), tRNA(Asn), and tRNA(Tyr) are located upstream of the terminal three exons c, d, and e. The genomic distance, the interspersed tRNA genes, and the group II intron sequences flanking the two separated exons suggest trans-splicing to be required to connect exons b and c. Maturation of the mRNA includes RNA editing at 36 sites in the open reading frame. Three RNA editing events are observed in the split group II intron sequences. Two of these events allow after editing additional base pairings in the secondary structure, one in the stem of domain I, the other in the putative trans-pairing region of domain IV. These RNA editings may thus be involved in the trans-splicing reaction.

Amino Acid Sequence↗
PubMed2000

Alternative design of a tRNA core for aminoacylation.

The core of Escherichia coli tRNA(Cys) is important for aminoacylation of the tRNA by cysteine-tRNA synthetase. This core differs from the common tRNA core by having a G15:G48, rather than a G15:C48 base-pair. Substitution of G15:G48 with G15:C48 decreases the catalytic efficiency of aminoacylation by two orders of magnitude. This indicates that the design of the core is not compatible with G15:C48. However, the core of E. coli tRNA(Gln), which contains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided by the core of E. coli tRNA(Gln), we sought to test and identify alternative functional design of the tRNA(Cys) core that contains G15:C48. Although analysis of the crystal structure of tRNA(Cys) and tRNA(Gln) implicated long-range tertiary base-pairs above and below G15:G48 as important for a functional core, we showed that this was not the case. The replacement of tertiary interactions involving 9, 21, and 59 in tRNA(Cys) with those in tRNA(Gln) did not construct a functional core that contained G15:C48. In contrast, substitution of nucleotides in the variable loop adjacent to 48 of the 15:48 base-pair created functional cores. Modeling studies of a functional core suggests that the re-constructed core arose from enhanced stacking interactions that compensated for the disruption caused by the G15:C48 base-pair. The repacked tRNA core displayed features that were distinct from those of the wild-type and provided evidence that stacking interactions are alternative means than long-range tertiary base-pairs to a functional core for aminoacylation.

Acylation↗
PubMed1992

The anticodon and discriminator base are major determinants of cysteine tRNA identity in vivo.

Mutants of the Escherichia coli initiator tRNA (tRNA(fMet)) have been used to examine the role of the anticodon and discriminator base in in vivo aminoacylation of tRNAs by cysteinyl-tRNA synthetase. Substitution of the methionine anticodon CAU with the cysteine anticodon GCA was found to allow initiation of protein synthesis by the mutant tRNA from a complementary initiation codon in a reporter protein. Sequencing of the protein revealed that cysteine comprised about half of the amino acid at the N terminus. An additional mutation, converting the discriminator base of tRNA(GCAfMet) from A73 to the base present in tRNA(Cys) (U73), resulted in a 6-fold increase in the amount of protein produced and insertion of greater than or equal to 90% cysteine in response to the complementary initiation codon. Substitution of C73 or G73 at the discriminator position led to insertion of little or no cysteine, indicating the importance of U73 for recognition of the tRNA by cysteinyl-tRNA synthetase. Single base changes in the anticodon of tRNA(GCAfMet) containing U73 from GCA to UCA, GUA, GCC, and GCG (changes underlined) eliminated or dramatically reduced cysteine insertion by the mutant initiator tRNA indicating that all three cysteine anticodon bases are essential for specific aminoacylation of the tRNA with cysteine in vivo.

Acylation↗
PubMed1993

Characterization of iron (II).bleomycin-mediated RNA strand scission.

The ability of iron(II).bleomycin to mediate RNA degradation was further characterized. At micromolar concentrations, FeII.BLM was shown to effect cleavage of Escherichia coli tRNA(1His) and a Schizosaccharomyces pombe amber suppressor tRNA construct in an efficient fashion. In contrast, E. coli tRNA(Cys) and yeast mitochondrial tRNA(Asp) and tRNA(fMet) precursors were not substrates for FeII.BLM. Also shown to be a good substrate for cleavage by FeII.BLM was yeast 5S ribosomal RNA. Since HIV-1 reverse transcriptase mRNA has previously been shown to be degraded by Fe.BLM (Carter et al., 1990a), members of the three major classes of RNA have now been shown to undergo Fe.BLM-mediated strand scission. For each of the substrate RNAs, cleavage occurred at sites unique to that substrate. Although RNA cleavage occurred at numerous sequences, 5'-G-pyr-3' sites were prominent. Likewise, while cleavage was noted in regions anticipated to be double-stranded, as well as in single-stranded regions, a disproportionate number of cleavages were noted at the junction between single- and double-stranded regions. As found in earlier studies, RNA cleavage was much more selective than DNA cleavage. Further, when RNA cleavage was carried out in the presence of reagents such as Mg2+, spermidine, and NaCl, the selectivity of cleavage was further enhanced. The highly selective and efficient cleavage of a number of RNA molecules reinforces our earlier suggestion that RNA may constitute a therapeutically relevant target for bleomycin.

Base Sequence↗
PubMed1994

Dual hydrolytic role for Pb(II) ions.

RNA phosphodiester bonds can be cleaved by metal ions, of which Pb2+ is one of the most effective. It can cleave both generally and site-specifically, depending on the substrate and the conditions. In addition, metal ions are also known to cleave ester bonds between amino acid and the 3'-end of transfer RNA. Here we report that in aminoacylated transfer RNA, Pb2+ ions cleave internucleotide bonds in the 3'-end of tRNA and also cleaves the bond between tRNA and its amino-acid, attached at the 3'-end via an ester bond to the terminal ribose in aminoacyl tRNA. The two reactions proceed at different rates. The rate of deacylation is significantly faster than the rate of cleavage of phosphodiester bonds, with a pH-optimum of 7. This dual hydrolytic role is not seen for other metal ions examined, namely Zn(II), Cd(II) and Mn(II). The rate of the two kinds of hydrolyses by Pb2+ ions is compared with that of other metal-ions. The mechanism of cleavage is investigated further by modification of the 3'-end of tRNA.

Acylation↗
PubMed1994

Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy.

We have investigated nine children with infantile onset of mitochondrial myopathy and two adults with myoclonus epilepsy and ragged-red fibers (MERRF) and chronic progressive external ophthalmoplegia (CPEO), respectively. These patients lacked any of the previously known pathogenic tRNA mutations. Southern blot analysis of muscle mtDNA revealed no deletions. The tRNA genes of muscle mtDNA were sequenced. Restriction enzyme analysis of PCR fragments was performed to verify the presence of the mutations identified by automatic sequencing. Several tRNA mutations were found, but they were all homoplasmic. Furthermore, the mutations were either present in controls or did not change nucleotides conserved between species. This strongly suggests that none of the tRNA mutations identified in the 11 patients with mitochondrial encephalomyopathy was pathogenic. It can thus be concluded that mitochondrial tRNA mutations and mtDNA deletions probably are an infrequent cause of mitochondrial disorders in infants. Patients with MERRF and CPEO may lack both pathogenic point mutations of tRNA genes and deletions of mtDNA.

Adult↗
PubMed1998

5HT2C CYS23/SER23 polymorphism is not associated with obsessive-compulsive disorder.

A great deal of evidence suggests that a genetic component underlies obsessive-compulsive disorder (OCD). The response to serotonergic medications and the worsening of obsessive symptoms after administration of serotonergic agonists indicate that serotonergic mechanisms are involved in OCD. We investigated the role of the Cys23Ser mutation of the 5HT2C receptor gene in the etiology of this disorder by performing an association study comparing a sample of 109 OCD patients with a sample of 107 healthy control subjects. No allelic or genotypic association of OCD with the 5HT2C receptor gene mutation was revealed in our data. We also extended the association analysis to a subsample of 39 OCD patients that had previously been submitted to a challenge test with clomipramine. In the subsample of OCD patients that received the challenge with clomipramine, no association between the 5HT2C receptor gene mutation and response to the challenge test was found. Our results exclude any specific role of the Cys23Ser mutation of 5HT2C receptor gene in the etiology of OCD: it seems probable that more complex genetic models are needed to explain the involvement of serotonergic elements in the etiology of this disorder.

Adult↗
PubMed1997

An RNA structural determinant for tRNA recognition.

Escherichia coli tRNACys contains an unusual G15.G48 tertiary base pair that is important for recognition and aminoacylation by cysteine tRNA synthetase. This G15.G48 tertiary base pair has a distinctive chemical modification signature that suggests an N2.N3 base pairing. The N2.N3 pairing of a G.G base pair has not been described in any existing RNA structures. Identification of the structural determinant of G15.G48 is of fundamental importance for understanding the formation of an RNA tertiary base pair, as well as the role of RNA tertiary structure in tRNA recognition. We show here that the structural determinant for G15.G48 is an A13.A22 mismatch in the dihydrouridine stem. Introduction of A13.A22 to an unrelated tRNA confers the distinctive chemical modification signature of G15. G48 while substitution of A13.A22 eliminates this signature. The relationship between G15.G48 and A13.A22 enables the unrelated tRNA to be efficiently recognized by cysteine tRNA synthetase. Modeling studies show that A13.A22 has the potential to form a base triple with A46, which is directly connected to G48 in the G15.G48 base pair. The proposed A13.A22.A46 base triple provides a framework for understanding how two RNA structural elements may be related to each other in playing an important role in tRNA aminoacylation.

Acylation↗
PubMed1998

Processing and editing of overlapping tRNAs in human mitochondria.

Overlapping tRNA genes in mitochondria of many metazoans introduce a problem for the processing of such polycistronic primary transcripts. Using runoff transcripts and an S100 extract from HeLa cell mitochondria, the processing of the human mitochondrial tRNATyr/tRNACys precursor (carrying an overlap of one base) was investigated: tRNACys is released in its complete form carrying the overlapping residue at the first position, whereas tRNATyr lacks that nucleotide at the discriminator position. Partial deletion of tRNACys or complete replacement by a non-tRNA-like sequence does not alter the processing reaction and indicates that the upstream tRNATyr alone is recognized by a 3'-endonuclease activity. The truncated 3'-end of this tRNATyr is then completed in an editing reaction that incorporates the missing residue. The processing of this tRNA overlap seems to be species-specific, because an overlapping tRNA precursor (tRNASer(AGY)/tRNALeu(CUN)) from opossum mitochondria is not recognized by the human extract. Because processing activities for overlapping and nonoverlapping tRNA precursors could not be separated, it seems that one general activity is responsible for the 3'-end processing of mitochondrial tRNAs and that this activity coevolved with the particular overlap between tRNATyr and tRNACys in human mitochondria, being unable to recognize overlaps between other tRNAs.

Base Sequence↗