Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “tRNA”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources
PubMed1990

Recognition of tRNA(Tyr) by tyrosyl-tRNA synthetase.

In this review, I have brought together and compared the available data on the interaction between tRNA(Tyr) and tyrosyl-tRNA synthetases (TyrTS) of prokaryotic origins. The amino acid sequences of the heterologous TyrTS that can charge Escherichia coli tRNA(Tyr), show that the residues involved in the binding and recognition of tyrosine are strictly conserved whereas those involved in the interaction with tRNA(Tyr) are only weakly similar. The results of in vivo genetic complementation experiments indicate that the identity elements of tRNAs and the recognition mechanisms of such elements by the synthetases have been conserved during evolution. Heterologous or mutant tRNA(Tyr) are quantitatively charged by E coli TyrTS; the set of their common residues contains less than 10 elements if one excludes the invariant and semi-invariant residues of tRNAs. The residues of this set are candidates for a specific recognition by TyrTS. So far, adenosine-73 is the only residue for which a specific recognition of the base has been demonstrated. The residues that might serve as identity elements for E coli tRNA(Tyr) [McClain WH, Nicholas Jr HB (1987) J Mol Biol 194, 635-642] do not belong to the above set of conserved residues and therefore probably play negative roles, enabling tRNA(Tyr) to avoid non-cognate synthetases. Comparison of the charging and stability properties of mutant tRNA(Tyr) su +3 shows that bases 1 and 72 must pair (either by Watson-Crick or non-canonical hydrogen bonds) and adopt a geometry which is compatible with the helical structure of the acceptor stem in order for the mutant tRNA(Tyr) to be charged with tyrosine. If bases 1 and 72 or bases 2 and 71 cannot form such pairings, the suppressor phenotype of the mutant tRNA(Tyr)su +3 becomes thermosensitive. The weakening of base pair 1/72 by mutation or the change of adenosine-73 into guanosine results in the charging of tRNA(Tyr)su +3 with glutamine. Comparison of the structural model of the TyrTS/tRNA(Tyr) complex with the crystallographic structure of the GlnTS/tRNA(Gln) complex indicates that the mechanisms for the recognition of the acceptor arm are different in the 2 cases. Chemical attack and molecular modeling experiments have indicated that the acceptor end of tRNA(Tyr) ... CCCA3'-OH, remains mobile after the initial binding of tRNA(Tyr) to TyrTS.

Base Sequence↗
PubMed2001

The appended C-domain of human methionyl-tRNA synthetase has a tRNA-sequestering function.

An ancillary RNA-binding domain is appended to the C-terminus of human methionyl-tRNA synthetase. It comprises a helix-turn-helix (HTH) motif related to the repeated units of the linker region of bifunctional glutamyl-prolyl-tRNA synthetase, and a specific C-terminal KGKKKK lysine-rich cluster (LRC). Here we show by gel retardation and tRNA aminoacylation experiments that these two regions are important for tRNA binding. However, the two pieces of this bipartite RNA-binding domain are functionally distinct. Analysis of MetRS mutant enzymes revealed that the HTH motif is more specifically endowed with a tRNA-sequestering activity and confers on MetRS a rate-limiting dissociation of aminoacylated tRNA. Elongation factor EF-1alpha enhanced the turnover in the aminoacylation reaction. In contrast, the LRC region is most probably involved in accelerating the association step of deacylated tRNA. These two nonredundant RNA-binding motifs strengthen tRNA binding by the synthetase. The native form of MetRS, containing the C-terminal RNA-binding domain, behaves as a processive enzyme; release of the reaction product is not spontaneous, but may be synchronized with the subsequent step of the tRNA cycle through EF-1alpha-assisted dissociation of Met-tRNA(Met). Therefore, the eukaryotic-specific C-domain of human MetRS may have a dual function. It may ensure an efficient capture of tRNA(Met) under conditions of suboptimal deacylated tRNA concentration prevailing in vivo, and may instigate direct transfer of aminoacylated tRNA from the synthetase to elongation factor EF-1alpha.

Amino Acid Sequence↗
PubMed1991

Structural similarities in glutaminyl- and methionyl-tRNA synthetases suggest a common overall orientation of tRNA binding.

Detailed comparisons between the structures of the tRNA-bound Escherichia coli glutaminyl-tRNA (Gln-tRNA) synthetase [L-glutamine:tRNA(Gln) ligase (AMP-forming), EC 6.1.1.18] and recently refined E. coli methionyl-tRNA (Met-tRNA) synthetase [L-methionine:tRNA(Met) ligase (AMP-forming), EC 6.1.1.10] reveal significant similarities beyond the anticipated correspondence of their respective dinucleotide-fold domains. One similarity comprises a 23-amino acid alpha-helix-turn-beta-strand motif found in each enzyme within a domain that is inserted between the two halves of the dinucleotide binding fold. A second correspondence, which consists of two alpha-helices connected by a large loop and beta-strand, is located in the Gln-tRNA synthetase within a region that binds the inside corner of the "L"-shaped tRNA molecule. This structural motif contains a long alpha-helix, which extends along the entire length of the D and anticodon stems of the complexed tRNA. We suggest that the positioning of this helix relative to the dinucleotide fold plays a critical role in ensuring the proper global orientation of tRNA(Gln) on the surface of the enzyme. The structural correspondences suggest a similar overall orientation of binding of tRNA(Met) and tRNA(Gln) to their respective synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1988

Valyl-tRNA synthetase gene of Escherichia coli K12. Primary structure and homology within a family of aminoacyl-TRNA synthetases.

The DNA nucleotide sequence of the valS gene encoding valyl-tRNA synthetase of Escherichia coli has been determined. The deduced primary structure of valyl-tRNA synthetase was compared to the primary sequences of the known aminoacyl-tRNA synthetases of yeast and bacteria. Significant homology was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. In pairwise comparisons the highest level of homology was detected between the homologous valyl-tRNA synthetases of yeast and E. coli, with an observed 41% direct identity overall. Comparisons between the valyl- and isoleucyl-tRNA synthetases of E. coli yielded the highest level of homology detected between heterologous enzymes (19.2% direct identity overall). An alignment is presented between the three branched-chain aminoacyl-tRNA synthetases (valyl- and isoleucyl-tRNA synthetases of E. coli and yeast mitochondrial leucyl-tRNA synthetase) illustrating the close relatedness of these enzymes. These results give credence to the supposition that the branched-chain aminoacyl-tRNA synthetases along with methionyl-tRNA synthetase form a family of genes within the aminoacyl-tRNA synthetases that evolved from a common ancestral progenitor gene.

Amino Acid Sequence↗
PubMed1993

Purification of aminoacyl-tRNA synthetase kinase activities associated with threonyl- and tyrosyl-tRNA synthetases isolated from Bom:NMRI mouse liver.

Aminoacyl-tRNA synthetase kinase activities and aminoacyl-tRNA synthetase activities prepared from postribosomal supernatants of Bom:NMRI mouse liver comigrated through the following purification steps: 1 a) gel filtration on Sephadex G-200, 1 b) chromatofocusing, 2) affinity chromatography on immobilised total tRNA (mixture of tRNA's, specific for all aminoacyl-tRNA synthetases) and 3) affinity chromatography on immobilised tRNA, specific for each of threonyl- and tyrosyl-tRNA synthetases. The purification factors for threonyl- and tyrosyl-tRNA synthetases were about 17000x. The purified synthetases showed only one protein band following sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified threonyl- and tyrosyl-tRNA synthetase proteins were found also to possess threonyl- and tyrosyl-tRNA synthetase kinase activities, respectively. The purification of tRNA(Thr) and tRNA(Tyr) as well as a method for the renaturation and identification of threonyl- and tyrosyl-tRNA synthetase activities in protein bands obtained by SDS PAGE are described.

Animals↗
PubMed1993

Recognition nucleotides of Escherichia coli tRNA(Leu) and its elements facilitating discrimination from tRNASer and tRNA(Tyr).

In order to study how Escherichia coli leucyl-tRNA synthetase recognizes tRNA(Leu) and discriminates it from the other two class II tRNAs, tRNA(Ser) and tRNA(Tyr), various mutations were introduced into class II tRNA transcripts. The discriminator base A73, but not the anticodon sequence, was found to serve as a critical recognition element of tRNA(Leu). A base substitution at the invariant nucleotide A14, but not at any of the other nucleotides characteristic of the E. coli tRNA(Leu) isoacceptors among the three class II tRNAs, caused significantly damaged aminoacylation with leucine. A two base-pair deletion in the long variable arm also resulted in no significant decrease of activity. Transplanting the three tertiary elements characteristic of E. coli tRNA(Leu) (i.e. the location of the G18G19 sequence in the D-loop, the A15 U48 base-pair and the stem pairing pattern of the long variable arm) besides the discriminator base change introduced the leucine charging activity in terms of Vmax/Km, up to 0.1 of that for the normal sequence of tRNA(Leu) into both tRNA(Ser) and tRNA(Tyr). These results indicate that A73 and A14 (or its vicinity) are involved in recognition by leucyl-tRNA synthetase, and that several tertiary elements play a significant role in the discrimination of tRNA(Leu) from the other two class II tRNAs.

Base Sequence↗
PubMed1992

Synthetase competition and tRNA context determine the in vivo identify of tRNA discriminator mutants.

The discriminator nucleotide (position 73) in tRNA has long been thought to play a role in tRNA identity as it is the only variable single-stranded nucleotide that is found near the site of aminoacylation. For this reason, a complete mutagenic analysis of the discriminator in three Escherichia coli amber suppressor tRNA backgrounds was undertaken; supE and supE-G1C72 glutamine tRNAs, gluA glutamate tRNA and supF tyrosine tRNA. The effect of mutation of the discriminator base on the identity of these tRNAs in vivo was assayed by N-terminal protein sequencing of E. coli dihydrofolate reductase, which is the product of suppression by the mutated amber suppressors, and confirmed by amino acid specific suppression experiments. In addition, suppressor efficiency assays were used to estimate the efficiency of aminoacylation in vivo. Our results indicate that the supE glutamine tRNA context can tolerate multiple mutations (including mutation of the discriminator and first base-pair) and still remain predominantly glutamine-accepting. Discriminator mutants of gluA glutamate tRNA exhibit increased and altered specificity probably due to the reduced ability of other synthetases to compete with glutamyl-tRNA synthetase. In the course of these experiments, a glutamate-specific mutant amber suppressor, gluA-A73, was created. Finally, in the case of supF tyrosine tRNA, the discriminator is an important identity element with partial to complete loss of tyrosine specificity resulting from mutation at this position. It is clear from these experiments that it may not be possible to assign a specific role in tRNA identity to the discriminator. The identity of a tRNA in vivo is determined by competition among aminoacyl-tRNA synthetases, which is in turn modulated by the nucleotide substitution as well as the tRNA context.

Amino Acids↗
PubMed1981

Comparison of the rate of aminoacylation of tRNA isolated from NMRI mouse liver with tRNA isolated from Krebs II ascites or mouse plasmacytoma cells.

A comparison of the initial rates of aminoacylation of tRNAs isolated from different sources for 17 amino acids was performed. tRNA was isolated from NMRI mouse liver (tRNA L) and from Krebs II ascites tumors (tRNA Asc), and aminoacyl-tRNA synthetases were prepared from the latter cells. The aminoacylation of tRNA Asc was 31-88% slower than the charging of tRNA L. In similar studies, tRNA from a mouse plasmacytoma tumor (tRNA Mt) and from suspension cultured cells of the same cell line (tRNA M) were compared to tRNA L in the aminoacylation reaction catalysed by synthetases isolated from tumor or suspension cultured cells. About half of the tRNAs (Mt or M) for the 17 amino acids tested differed in charging rate when compared to tRNA L, but the differences were not as great as those observed in the experiments where tRNA Asc and tRNA L were compared.

Amino Acyl-tRNA Synthetases↗
PubMed1998

A peculiarity of the reaction of tRNA aminoacylation catalyzed by phenylalanyl-tRNA synthetase from the extreme thermophile Thermus thermophilus.

It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low: the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNAPhe but also Escherichia coli tRNAPhe and E. coli tRNAPhe transcript, indicating that the presence of modified nucleotides is not necessary for tRNAPhe overcharging. Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases. Aminoacylated tRNAPhe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed.

Acylation↗
PubMed2005

tRNA-dependent aminoacyl-adenylate hydrolysis by a nonediting class I aminoacyl-tRNA synthetase.

Glutaminyl-tRNA synthetase generates Gln-tRNA(Gln) 10(7)-fold more efficiently than Glu-tRNA(Gln) and requires tRNA to synthesize the activated aminoacyl adenylate in the first step of the reaction. To examine the role of tRNA in amino acid activation more closely, several assays employing a tRNA analog in which the 2'-OH group at the 3'-terminal A76 nucleotide is replaced with hydrogen (tRNA(2'HGln)) were developed. These experiments revealed a 10(4)-fold reduction in kcat/Km in the presence of the analog, suggesting a direct catalytic role for tRNA in the activation reaction. The catalytic importance of the A76 2'-OH group in aminoacylation mirrors a similar role for this moiety that has recently been demonstrated during peptidyl transfer on the ribosome. Unexpectedly, tracking of Gln-AMP formation utilizing an alpha-32P-labeled ATP substrate in the presence of tRNA(2'HGln) showed that AMP accumulates 5-fold more rapidly than Gln-AMP. A cold-trapping experiment revealed that the nonenzymatic rate of Gln-AMP hydrolysis is too slow to account for the rapid AMP formation; hence, the hydrolysis of Gln-AMP to form glutamine and AMP must be directly catalyzed by the GlnRS x tRNA(2'HGln) complex. This hydrolysis of glutaminyl adenylate represents a novel reaction that is directly analogous to the pre-transfer editing hydrolysis of noncognate aminoacyl adenylates by editing synthetases such as isoleucyl-tRNA synthetase. Because glutaminyl-tRNA synthetase does not possess a spatially separate editing domain, these data demonstrate that a pre-transfer editing-like reaction can occur within the synthetic site of a class I tRNA synthetase.

Adenosine Monophosphate↗
PubMed2005

Aspartyl-tRNA synthetase requires a conserved proline in the anticodon-binding loop for tRNA(Asn) recognition in vivo.

Most prokaryotes require Asp-tRNA(Asn) for the synthesis of Asn-tRNA(Asn). This misacylated tRNA species is synthesized by a non-discriminating aspartyl-tRNA synthetase (AspRS) that acylates both tRNA(Asp) and tRNA(Asn) with aspartate. In contrast, a discriminating AspRS forms only Asp-tRNA(Asp). Here we show that a conserved proline (position 77) in the L1 loop of the non-discriminating Deinococcus radiodurans AspRS2 is required for tRNA(Asn) recognition in vivo. Escherichia coli trpA34 was transformed with DNA from a library of D. radiodurans aspS2 genes with a randomized codon 77 and then subjected to in vivo selection for Asp-tRNA(Asn) formation by growth in minimal medium. Only proline codons were found at position 77 in the aspS2 genes isolated from 21 of the resulting viable colonies. However, when the aspS temperature-sensitive E. coli strain CS89 was transformed with the same DNA library and then screened for Asp-tRNA(Asp) formation in vivo by growth at the non-permissive temperature, codons for seven other amino acids besides proline were identified at position 77 in the isolates examined. Thus, replacement of proline 77 by cysteine, isoleucine, leucine, lysine, phenylalanine, serine, or valine resulted in mutant D. radiodurans AspRS2 enzymes still capable of forming Asp-tRNA(Asp) but unable to recognize tRNA(Asn). This strongly suggests that proline 77 is responsible for the non-discriminatory tRNA recognition properties of this enzyme.

Acylation↗
PubMed1992

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).

Animals↗
PubMed1996

An example of non-conservation of oligomeric structure in prokaryotic aminoacyl-tRNA synthetases. Biochemical and structural properties of glycyl-tRNA synthetase from Thermus thermophilus.

Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D.T., Mazauric, M.-H., Kern, D. & Moras, D. (1995) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha 2 type consistent with that found for eukaryotic Gly-tRNA synthetases (yeast and Bombyx mori), but different from that of Gly-tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are alpha 2 beta 2 tetramers. N-terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenzae and Coxiella burnetii) albeit the enzyme is deprived of the N-terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly-tRNA synthetase combines strong structural homologies of eukaryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-PPi exchange and aminoacylation activities up to 70 degrees C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA(Gly) does not induce protection. Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA(gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(GCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity. The results are discussed in the context of the three-dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.

Amino Acid Sequence↗
PubMed1989

Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution.

The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences. The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1. Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA. The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures. Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3; (ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences. The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is

Adenosine Triphosphate↗
PubMed1976

Mechanism of aminoacylation of tRNA. Proof of the aminoacyl adenylate pathway for the isoleucyl- and tyrosyl-tRNA synthetases from Escherichia coli K12.

The following observations show that the formation of isoleucyl-tRNA catalyzed by the isoleucyl-tRNA synthetase from Escherichia coli K12 involves the initial rapid formation of an isoleucyl adenylate complex followed by the slow, rate-determining, transfer of the isoleucyl moiety to tRNA. (1) The rate constant for the transfer of [14C]Ile from the E-[14C]Ile approximately AMP complex to tRNA is the same as the turnover number for the steady-state isoleucylation of tRNA at pH 7.78 (1.5 s-1) and pH 5.87 (0.34 s-1). (2) On mixing a solution of isoleucyl-tRNA synthetase and tRNA with [14C]Ile and ATP the steady-state rate of isoleucylation is attained in the first turnover of the enzyme, with little or no "burst" or charging that would indicate a slow step after the transfer step. (3) The pyrophosphate exchange reaction in the presence of tRNA is 40 times faster than the overall rate of isoleucylation of tRNA. (4) Similarly, rapid quenching experiments indicate that isoleucyl adenylate is formed prior to the transfer step. The possibility that isoleucyl adenylate formation is a parallel reaction caused by a second active site on the enzyme is ruled out both by the stoichiometry in this rapid quenching experiment and also the overall stoichiometry of isoleucyl-tRNA formation. At saturating reagent concentrations the major species in solution is the E-tRNA-Ile approximately AMP complex. Similar observations are found for the tyrosyl-tRNA systhetase except that at saturating reagent concentrations the rate constants for both tyrosyl adenylate formation and transfer are similar so that both processes contribute to the rate-determining step.

Adenosine Monophosphate↗
PubMed1993

Solution structure of selenocysteine-inserting tRNA(Sec) from Escherichia coli. Comparison with canonical tRNA(Ser).

Selenocysteine-inserting tRNAs (or tRNA(Sec)) are structurally untypical tRNAs that are charged by seryl-tRNA synthetase before being recognized by the selenocysteine synthase that converts serine into selenocysteine. tRNA(Sec) from Escherichia coli contains 95 nucleotides and is the longest tRNA known to date, in contrast to canonical tRNA(Ser), 88 nucleotides-long. We have studied its solution conformation by chemical and enzymatic probing. Global structural features were obtained by cobra venom and S1 nuclease mapping, as well as by probing with Pb2+. Accessibilities of phosphate groups were measured by ethylnitrosourea probing. Information about positions in bases involved in Watson-Crick pairing, in stacking or in tertiary interactions were obtained by chemical probing with dimethylsulfate, diethylpyrocarbonate, kethoxal and carbodiimide. On the basis of these chemical data, a three-dimensional model was constructed by computer modeling and compared to that of canonical tRNA(Ser). tRNA(Sec) resembles tRNA(Ser) at the level of its T-arm and anticodon-arm conformations, as well as at the joining of the D- and T-loops by a tertiary Watson-Crick G19-C56 interaction. Its extra-long variable arm is a double-stranded structure closed by a four nucleotide loop that is linked to the body of the tRNA in a way different from that found in tRNA(Ser). As anticipated from the peculiar features of the sequence in the D-loop and at the junction of amino acid and D-arms, tRNA(Sec) possesses a novel but restricted set of tertiary interactions in the core of its three-dimensional structure: a G8-A21-U14 triple pair and a novel interaction between C16 of the D-loop and C59 of the T-loop. A third triple interaction involving C15-G20a-G48 is suggested but some experimental evidence for it is still lacking. It is furthermore concluded that the D-arm has six base-pairs instead of three, as in canonical class II tRNA(Ser), with the D-loop containing only four nucleotides. Finally, the amino acid accepting arm forms a stack of eight Watson-Crick base-pairs (instead of 7 in other tRNAs). The biological relevance of this model with regard to interaction with seryl-tRNA synthetase and enzymes from the selenocysteine metabolism is discussed.

Adenine↗
PubMed1987

Homology in the region containing a tRNA(Trp) gene and a (complete or partial) tRNA(Pro) gene in wheat mitochondrial and chloroplast genomes.

We have used bean mitochondrial (mt) and chloroplast (cp) tRNA(Trp) as probes to locate the corresponding genes on the mt and cp genomes of wheat and we have determined the nucleotide sequences of the wheat mt and cp tRNA(Trp) genes and of the flanking regions. Sequence comparisons show that the wheat mt and cp tRNA(Trp) genes are 97% homologous. On the wheat cp DNA, a tRNA(UGGPro) gene was found 139 bp upstream of the cp tRNA(Trp) gene. On the wheat mt DNA, a sequence of 23 nucleotides completely homologous with the 3' end of this cp tRNA(Pro) gene was found 136 bp upstream of the mt tRNA(Trp) gene, but there is only 38% homology between cp and mt wheat genomes in the intergenic regions. The overall organization of this region in the chloroplast genome (a tRNA(Trp) gene separated by about 140 bp from a tRNA(Pro) gene) is also found in the mitochondrial genome, suggesting that this mitochondrial fragment might have originated from a chloroplast DNA insertion. A comparison of the genes and of the intergenic regions located between the tRNA(Trp) gene and the tRNA(Pro) (or partial tRNA(Pro)) gene shows that there is an almost complete conservation of these sequences in the mitochondrial DNA of wheat and maize, whereas wheat mt and cp intergenic regions show more sequence divergence. Wheat mt tRNA(Trp) gene is encoded by the main mt genome (accounted for by the master chromosome) but, in the case of maize mitochondria, this gene was found to be encoded by the 2.3 kb linear plasmid, indicating that this plasmid is not dispensable in maize mitochondria.

Base Sequence↗
PubMed1992

Expression of variant nuclear Arabidopsis tRNA(Ser) genes and pre-tRNA maturation differ in HeLa, yeast and wheat germ extracts.

We have recently identified a tRNA gene cluster in the Arabidopsis nuclear genome. One tRNA(Ser) (AGA) gene and two tRNA(Tyr) (GTA) genes occur in tandem arrangement on a 1.5 kb unit that is amplified about 20-fold at a single chromosomal site. Here we have studied the in vitro expression of seven individually cloned tRNA(Ser) genes (pAtS1 to pAtS7) derived from this cluster. Five out of the seven tRNA(Ser) genes contain point mutations in the coding region which have in part adverse effects on the expression of these genes in different cell-free systems: (i) C10 and A62 in plant tRNA(Ser) genes, which correspond to G10 and C62, respectively, in all known vertebrate tRNA genes, result in a reduced transcription efficiency in HeLa but not in yeast extract. This indicates that yeast RNA polymerase III tolerates nucleotide substitutions at positions 10 [5' internal control region (ICR)] and 62 (3' ICR), whereas the vertebrate RNA polymerase III requires a more stringent consensus sequence. (ii) Processing of a pre-tRNA(Ser) with a mismatch in the aminoacyl stem is impaired in HeLa, yeast and wheat germ extracts, however, a mismatch in the anticodon stem is deleterious for HeLa and wheat germ but not for yeast processing enzymes. The unexpectedly high number of potential tRNA(Ser) pseudogenes in the cluster - quite in contrast to the tRNA(Ser) genes which mainly code for functional tRNAs - suggested that tRNA(Ser) (AGA) genes also occur elsewhere in the genome. We present evidence that single copies of tRNA(Ser) (AGA) genes do indeed exist outside the tRNA gene cluster.

Base Sequence↗