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At least 73 records · Page 4Linked to original sources
PubMed1972

Phenylalanyl-tRNA synthetase and isoleucyl-tRNA Phe : a possible verification mechanism for aminoacyl-tRNA.

The synthesis of isoleucyl-tRNA(Phe) (Escherichia coli) proceeds at an appreciable rate under normal in vitro conditions in the presence of isoleucyl-tRNA synthetase (EC 6.1.1.5) from E. coli. The misacylated product is shown here to be hydrolyzed by highly purified phenylalanyl-tRNA synthetase from E. coli, with release of isoleucine and active tRNA(Phe). Thus, phenylalanyl-tRNA synthetase possesses a previously unrecognized activity, which deacylates a mistakenly acylated tRNA(Phe); the enzyme is inactive toward correctly matched aminoacyl tRNAs. Such a mechanism could serve to verify aminoacyl-tRNAs, deacylating those that are misacylated. Thus, a common generalization needs to be modified: an amino acid is not necessarily committed to a given (incorrect) anticodon when it is incorporated into aminoacyl-tRNA. It may be possible to correct it thereafter.

Acylation↗
PubMed1996

The crystal structures of T. thermophilus lysyl-tRNA synthetase complexed with E. coli tRNA(Lys) and a T. thermophilus tRNA(Lys) transcript: anticodon recognition and conformational changes upon binding of a lysyl-adenylate analogue.

The crystal structures of Thermus thermophilus lysyl-tRNA synthetase, a class IIb aminoacyl-tRNA synthetase, complexed with Escherchia coli tRNA(Lys)(mnm5 s2UUU) at 2.75 A resolution and with a T. thermophilus tRNA(Lys)(CUU) transcript at 2.9 A resolution are described. In both complexes only the tRNA anticodon stem-loop is well ordered. The mode of binding of the anticodon stem-loop to the N-terminal beta-barrel domain is similar to that previously found for the homologous class IIb aspartyl-tRNA synthetase-tRNA(Asp) complex except in the region of the wobble base 34 where either mnm5 s2U or C can be accommodated. The specific recognition of the other anticodon bases, U-35 and U-36, which are both major identity elements in the lysine system, is also described. Additional crystallographic data on a ternary complex with a lysyl-adenylate analogue show that binding of the intermediate induces significant conformational changes in the vicinity of the active site of the enzyme.

Amino Acid Sequence↗
PubMed1997

Recognition of tRNA(Gly) by three widely diverged glycyl-tRNA synthetases: evolution of tRNA recognition.

Glycyl-tRNA synthetase (GlyRS) is an unusual aminoacyl-tRNA synthetase because it varies in its quarternary structure between organisms; Escherichia coli GlyRS is an alpha 2 beta 2 tetramer, whereas those of Thermus thermophilus and yeast are alpha 2 dimers. In contrast, the tRNA(Gly) sequence is virtually identical in E. coli and T. thermophilus but very different in yeast. In this study, we examined the molecular recognition of tRNA(Gly) by three widely diverged GlyRSs using in vitro tRNA transcripts. The results obtained in the mutation studies indicate that despite such large differences of the two prokaryotic GlyRSs, tRNA(Gly) identity has been essentially conserved in prokaryotes, and that there are also differences in the acceptor stem recognition between prokaryotes and yeast. The clear separation between prokaryotes and yeast is retained in the identity element location, whereas the apparent diversity of the two prokaryotic enzymes does not reflect on the tRNA recognition.

Base Sequence↗
PubMed1989

Sequence analysis of the tRNA(Tyr) and tRNA(Lys) genes and evidence for the transcription of a chloroplast-like tRNA(Met) in maize mitochondria.

The nucleotide sequences of three tRNA genes and their flanking regions from the maize mitochondrial genome is reported. These genes, which are located in the same region of the genome between the 14-kb inverted repeats, are transcribed in the mitochondria and code for tRNA(Lys) (anticodon UUU) tRNA(Met) (CAU) and tRNA(Tyr) (GUA). The very high homology shown by the tRNA(Met) gene with its chloroplast counterpart indicates that it probably originates from a chloroplast DNA insertion. The analysis of the upstream regions of these genes showed that the tRNA(Tyr) and the tRNA(Lys) genes possess the consensus sequence AAGAANRR, which could act as a promoter sequence in higher plant mitochondria.

Base Sequence↗
PubMed1996

Asn-tRNA in Lactobacillus bulgaricus is formed by asparaginylation of tRNA and not by transamidation of Asp-tRNA.

In many organisms (e.g., gram-positive eubacteria) Gin-tRNA is not formed by direct glutaminylation of tRNAGln but by a specific transamidation of Glu-tRNAGln. We wondered whether a similar transamidation pathway also operates in the formation of Asn-tRNA in these organisms. Therefore we tested in S-100 preparations of Lactobacillus bulgaricus, a gram-positive eubacterium, for the conversion by an amidotransferase of [14C]Asp-tRNA to [14C]Asn-tRNA. As no transamidation was observed, we searched for genes for asparaginyl-tRNA synthetase (AsnRS). Two DNA fragments (from different locations of the L.bulgaricus chromosome) were found each containing an ORF whose sequence resembled that of the Escherichia coli asnS gene. The derived amino acid sequences of the two ORFs (432 amino acids) were the same and 41% identical with E.coli AsnRS. When one of the ORFs was expressed in E.coli, it complemented the temperature sensitivity of an E.coli asnS mutant. S-100 preparations of this transformant showed increased charging of unfractionated L.bulgaricus tRNA with asparagine. Deletion of the 3'-terminal region of the L.bulgaricus AsnRS gene led to loss of its complementation and aminoacylation properties. This indicates that L.bulgaricus contains a functional AsnRS. Thus, the transamidation pathway operates only for Gin-tRNAGln formation in this organism, and possibly in all gram-positive eubacteria.

Amino Acid Sequence↗
PubMed1991

Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts.

A footprinting technique using phosphorothioate-containing RNA transcripts has been developed and applied to identify contacts between Escherichia coli tRNA(Ser) and its cognate aminoacyl-tRNA synthetase. The cloned gene for the tRNA was transcribed in four reactions in which a different NTP was complemented by 5% of the corresponding nucleoside 5'-O-(1-thiotriphosphate). The phosphorothioate groups of such transcripts are cleaved by reaction with iodine to permit sequencing of the transcripts. Footprinting was achieved by performing the same reaction with the phosphorothioate-tRNA-enzyme complex. At 1 mM iodine, selective protection of the tRNA transcripts in the cognate system was observed, with strong protection at positions 52 and 68 and weak protection at positions 46, 53, 67, 69, and 70. It is suggested that these regions of the tRNA interact with the helical arm of the synthetase.

Base Sequence↗
PubMed1989

On the non-linear Eadie plots of the tRNA kinetics and non-linear Dixon plots of the PPi inhibition kinetics of the aminoacyl-tRNA synthetases. An analysis of the aminoacylation of tRNA in a model reaction.

A model of the aminoacyl-tRNA synthetase reaction was analyzed by deriving a rate equation, and by calculating the aminoacylation rates at various values of the rate and equilibrium constants. The model specially contained the possibilities that (1) the activation of the amino acid occurs either with bound or non-bound tRNA, and that (2) the transfer of the aminoacyl moiety from the aminoacyl adenylate to tRNA occurs either with bound or non-bound PPi. The analysis showed that the Eadie plots (tRNA as the variable substrate) are straight lines only if the rates of the activation reactions with bound and non-bound tRNA are equal. Otherwise the Eadie plots can be either curved upwards or downwards. The Dixon plots of the PPi inhibition are straight lines only if PPi must be dissociated from the enzyme before the transfer reaction. The conditions under which the Kiapp values are much lower than the dissociation constants for PPi are met if the transfer reaction is relatively slow and the reverse reaction of the activation (pyrophosphorolysis) is fast, and if the tRNA concentration is low.

Acylation↗
PubMed2003

Specific inhibition of the synthesis of human lysyl-tRNA synthetase results in decreases in tRNA(Lys) incorporation, tRNA(3)(Lys) annealing to viral RNA, and viral infectivity in human immunodeficiency virus type 1.

The major human tRNA(Lys) isoacceptors, and, are selectively packaged into human immunodeficiency virus type 1 (HIV-1) during assembly, where acts as a primer for reverse transcription. Lysyl-tRNA synthetase (LysRS) is also incorporated into HIV-1, independently of tRNA(Lys), via its interaction with Gag, and is a strong candidate for being the signal that specifically targets tRNA(Lys) for viral incorporation. We have transfected 293T cells with HIV-1 proviral DNA and short interfering RNA (siRNA) specific for LysRS to study the effect of diminished cellular LysRS upon tRNA(Lys) packaging, annealing to viral genomic RNA, and viral production and infectivity. At early time points after siRNA transfection, an 80% inhibition of LysRS incorporation into viruses reflects an 80% reduction of newly synthesized LysRS, rather than a more limited 20 to 25% decrease in the concentration of total cell LysRS, indicating that newly synthesized LysRS in the cell may be the main source of viral LysRS. Viruses produced from cells transfected with siRNA show reduced tRNA(Lys) packaging, reduced annealing to viral RNA, and reduced viral infectivity.

HIV-1↗
PubMed1992

Structural requirements for tRNA methylation. Action of Escherichia coli tRNA(guanosine-1)methyltransferase on tRNA(1Leu) structural variants.

The Escherichia coli enzyme tRNA(m1G)methyltransferase, one of a group of post-transcription tRNA-modifying enzymes, shows remarkable specificity in selecting the tRNA species and the specific guanosine base to be methylated. To examine the structural basis of this specificity, we synthesized a total of 15 modifications of tRNA(1Leu) and measured their methylation reaction kinetics in vitro. Elimination of any one of the three tRNA side loops, the V loop, the T loop, or the D loop, reduced the Vmax for methylation by about 1 order of magnitude. Elimination of all three side loops reduced Vmax by about 2 orders of magnitude. Clearly, gross tRNA structure is important for full enzyme activity. At the bottom of the stem proximal to the anticodon loop, in the pair at positions 31-39, substitution of a G-C for a C-G, a change that should not weaken the helical structure, had little effect on Vmax or Km. However, substitution of a G for a C increased Vmax and Km, whereas substitution of a C for G sharply reduced Vmax and, to a lesser extent, Km. These results appear to be a consequence of the principle that purines are better than pyrimidines in the stacking of adjacent bases for stability. Stacking in the stem structure appears to be important for methylation enzyme activity. In the anticodon loop itself, changing a U to a C had little effect, but changing the G of the anticodon to a C reduced Vmax over 20-fold, demonstrating the importance of the presence of the anticodon G adjacent to the G being methylated for enzyme recognition.

Anticodon↗
PubMed1989

A new type of chemically modified tRNA as a tool for the study of tRNA-aminoacyl-tRNA synthetase interaction.

tRNA(Phe) in which the adenine and cytosine rings in the aminoacyl arm and in the anticodon loop were converted to alkylating derivatives by mild treatment with methyl chlorotetrolate was used to study the tRNA(Phe)-yeast phenylalanyl-tRNA(Phe) synthetase interaction. At neutral pH, modified tRNA inhibited the enzyme competitively. At pH 9 this binding is accompanied by irreversible inactivation of the enzyme due to alkylation of the alpha subunit of the synthetase. Such a derivatization of tRNA could probably be used to investigate the interaction of other tRNAs with their cognate synthetases.

Alkylation↗
PubMed1989

Arginyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2).

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10,000 and 60,000. In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role. Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Modification of one tRNA recognition site of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA].

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA results in a binding of 1 mole of the reagent per 1 mole of the enzyme. Exhaustive alkylation of phenylalanyl-tRNA synthetase completely blocks the aminoacylation and partially inhibits the reaction of ATP--[32P]pyrophosphate exchange. Removal of the tRNA moiety of the reagent by hydrolysis of the ester bond N-chlorambucilyl-phenylalanine and terminal adenosine does not result in a restoration of ATP--[32P]pyrophosphate exchange and aminoacylation activity. The latter result may testify a chemical modification of amino acid residues essential for enzymatic activity. Possibility of blocking one of the two tRNA binding sites is discussed.

Adenosine Triphosphate↗
PubMed1979

Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. IV. Mutants with increased levels of leucyl- or seryl-tRNA synthetase.

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase and seryl-tRNA synthetase were selected for growth at 40 degrees C. Among these, strains were found with increased levels of both thermolabile synthetases. Two distinct genetic loci were found responsible for enzyme overproduction. leuR, located near xyl, causes elevated levels of leucyl-tRNA synthetase; while serR, located near leu, causes elevated levels of seryl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Interaction of aminoacyl-tRNA with bacterial elongation factor Tu: GTP complex: effects of the amino group of amino acid esterified to tRNA, the amino acid side chain, and tRNA structure.

The present investigation was undertaken to see to what extent the alpha-amino group of the amino acid, the side chain of the amino acid of aminoacyl-tRNA, and the tRNA structure are involved in determining the affinity of aminoacyl-tRNA for bacterial elongation factor Tu-GTP complex. Various aminoacyl-tRNAs, mis-aminoacylated tRNAs, and formylated aminoacyl-tRNAs were prepared, and the dissociation constants of the ternary complexes of aminoacyl-tRNA with ET-Tu: GTP were determined by the RNase-resistance assay. The results indicated that the free amino-acid group of the amino acids in aminoacyl-tRNA is strongly required for binding with EF-Tu : GTP. In this concentration, the biological significance of formylation for Met-tRNAMetf species is discussed.

Amino Acids↗
PubMed1981

The evolution of multi-isoacceptor tRNA families. Sequence of tRNA Leu CAA and tRNA Leu CAG from Anacystis nidulans.

Two leucine tRNAs from the cyanophyte Anacystis nidulans have been isolated, and their complete nucleotide sequences have been determined by combining data from oligonucleotide fingerprints and sequencing gels. The two sequences are 87 nucleotides long, have the anticodons CAA and CAG, and differ from each other at a total of 28 positions. They have been compared to other known tRNA Leu sequences and incorporated into a phylogenetic tree comprising prokaryotic and chloroplastic tRNA Leu sequences. Mutations inferred from the tree show that some parts of the tRNA molecule are highly variable (the extra arm and the acceptor stem) while others are much more conserved (the D and T arms). The topology of the tree supports the idea that blue-green algae and chloroplasts share a common prokaryotic ancestor and show a basic divergence between XAA and XAG anticodon-containing tRNAs, suggesting that these two subfamilies result from an ancient gene duplication. Finally, comparison of this phylogenetic tree with those of other multi-isoacceptor tRNA families shows no common scheme, which may be due to independent refinement of codon-reading patterns in different tRNA families.

Base Sequence↗
PubMed1997

Nucleotide sequence of nuclear tRNA(Gly) genes and tRNA(Gly) pseudogenes from yellow lupin (Lupinus luteus): expression of the tRNA(Gly) genes in vitro and in vivo.

A nuclear DNA fragment (7.8 kb) from yellow lupin (L. luteus) was sequenced and shown to contain tRNA(Gly) (GGC) genes and tRNAGly (GGC) pseudogenes organized in three tandemly repeated units: of 2565 bp and 2564 bp, and one, truncated from its 3' end, of 1212 bp. Each unit contains an identical pair of a tRNA(Gly) gene and a pseudogene, both having the same polarity. The nucleotide sequence of the gene appears colinear to L. luteus cytoplasmic tRNA(Gly) (GGC) primary structure. All three genes are efficiently transcribed in HeLa-cell nuclear extract giving two primary transcripts. The main, longer primary transcripts have each an extremely long 3' trailer of about 100 nucleotides, the structure of which is specific only for tRNAGly genes and pseudogenes (80% homology) of the studied tandem (but not for other tRNA(Gly) genes of the yellow lupin genome) as it has been shown by Southern hybridization. This distinctive feature allowed to isolate putative tRNAGly precursor(s) encoded by at least one of the three tRNA(Gly) (GGC) genes from L. luteus seedlings.

Base Sequence↗
PubMed1989

Isoleucyl-tRNA synthetase from baker's yeast. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA; role of terminal hydroxyl groups aminoacylation of tRNA(Ile)-C-C-A.

Specificity with regard to amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA by isoleucyl-tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2 = 180-1700), indicating that at same amino acid concentrations isoleucine is 180-1700 times more attached to tRNA(Ile)-C-C-3'dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2 = 10,000-30,000). D2 values of the other amino acids are in the range of 2000-10,000. Recognition of most amino acids is achieved in a four-step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof-reading steps occur on the pre- and the post-transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post-transfer proof-reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post-transfer proof-reading step are lacking. The role of the terminal hydroxyl groups of the tRNA for post-transfer proof-reading is assigned to a simple neighbouring group effect. No preference for the 2' or 3' position in proof-reading can be postulated.

Amino Acyl-tRNA Synthetases↗
PubMed1997

tRNA recognition for modification: solution probing of tRNA complexed with Escherichia coli tRNA (guanosine-1) methyltransferase.

The interaction of Escherichia coli tRNA (guanosine-1) methyltransferase and tRNA(1Leu) transcripts has been probed using cleavage with iodine of phosphorothioate-substituted transcripts, lead acetate, and enzymes specific for single- and double-stranded RNA. All lytic agents protect the anticodon stem-loop and variable loop regions against cleavage, and some protection is also seen in core structures of the tRNA. Residues from both strands of the anticodon stem are protected against cleavage with iodine and lead by enzyme, yet positions G37 and G36, which are crucial for catalysis and binding, are not. This suggests that these residues may undergo structural perturbation in the presence of S-adenosyl methionine. Occupancy of the AdoMet site by the product S-adenosyl-homocysteine, a potent inhibitor of the enzyme, has little or no effect on tRNA binding or protection. Enhanced reactivity with lead is seen at residues located in the anticodon stem-loop, extra-loop, and core (C34, U47c, and G49), which suggests some perturbations in RNA structure might accompany binding.

Base Sequence↗