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At least 595 records · Page 33Linked to original sources
PubMed1998

Modeling the tertiary interactions in the eukaryotic selenocysteine tRNA.

A novel three-dimensional model of tertiary interactions in the core region of the eukaryotic selenocysteine tRNA is proposed based on the analysis of available nucleotide sequences. The model features the 7/5 tRNA(Sec) secondary structure characterized by seven and five base pairs in the acceptor and T-stems, respectively, and four nucleotides in the connector region between the acceptor and D-stems. The model suggests a unique system of tertiary interactions in the area between the major groove of the D-stem and the first base pair of the extra arm that provides a rigid orientation of the extra arm and contributes to the overall stability of the molecule. The model is consistent with available experimental data on serylation, selenylation, and phosphorylation of different tRNA(Sec) mutants. The important similarity between the proposed model and the structure of the tRNA(Ser) is shown. Based on this similarity, the ability of some tRNA(Ser) mutants to be serylated, selenylated, and phosphorylated was evaluated and found to be in a good agreement with experimental data.

Animals↗
PubMed2004

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNAs.

Elongation factor Tu (EF-Tu) binds GTP and aminoacyl-tRNA (aa-tRNA) forming a ternary complex which is delivered to the A-site of the ribosome. Animal mitochondrial tRNAs are quite unusual and lack many of the residues important for the stabilization of the structures of other tRNAs. The stabilities of the ternary complexes formed by mammalian mitochondrial and E. coli EF-Tu were determined with four bovine mitochondrial aa-tRNAs. The ternary complex with Phe-tRNA(Phe) has a Kd of about 75 nM. Equilibrium dissociation constants are tightest for the two native Ser-tRNA species (17 nM). Ternary complexes formed with the transcript of tRNA(AGY)Ser are 10-fold weaker than those formed with the native tRNA.

Journal Article↗
PubMed1995

Comparison of the enzymatic properties of the two Escherichia coli lysyl-tRNA synthetase species.

In Escherichia coli, lysyl-tRNA synthetase activity is encoded by either a constitutive lysS gene or an inducible one, lysU. The two corresponding enzymes could be purified at homogeneity from a delta lysU and a delta lysS strain, respectively. Comparison of the pure enzymes, LysS and LysU, indicates that, in the presence of saturating substrates, LysS is about twice more active than LysU in the ATP-PPi exchange as well as in the tRNALys aminoacylation reaction. Moreover, the dissociation constant of the LysU-lysine complex is 8-fold smaller than that of the LysS-lysine complex. In agreement with this difference, the activity of LysU is less sensitive than that of LysS to the addition of cadaverine, a decarboxylation product of lysine and a competitive inhibitor of lysine binding to its synthetase. This observation points to a possible useful role of LysU, under physiological conditions causing cadaverine accumulation in the bacterium. Remarkably, these conditions also induce lysU expression. Homogeneous LysU and LysS were also compared in Ap4A synthesis. LysU is only 2-fold more active than LysS in the production of this dinucleotide. This makes unlikely that the heat-inducible LysU species could be preferentially involved in the accumulation of Ap4A inside stressed Escherichia coli cells. This conclusion could be strengthened by determining the concentrations of Ap4N (N = A, C, G, or U) in a delta lysU as well as in a lysU+ strain, before and after a 1-h temperature shift at 48 degrees C. The measured concentration values were the same in both strains.

Acylation↗
PubMed1990

Yeast methionyl-tRNA synthetase: analysis of the N-terminal extension and the putative tRNA anticodon binding region by site-directed mutagenesis.

Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step. The Km for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration of the catalytic residues. The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.

Adenosine Triphosphate↗
PubMed1972

Growth-linked instability of a mutant valyl-transfer ribonucleic acid synthetase in Escherichia coli.

The valyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli strain NP2907, previously described as having an elevated K(m) for adenosine triphosphate and reduced stability in vitro compared to the wild type, was found to be conditionally thermolabile in vivo. The rate of inactivation of this enzyme at a particular temperature appears to be coordinated with the rate of growth; at 40 C this coordination results in equal rates of synthesis and destruction over a wide range of growth rates. In vitro studies showed that conditions favoring maintenance of the valyl-tRNA synthetase-valyl adenylate complex conferred complete protection against inactivation at 40 C, whereas the further addition of uncharged tRNA caused rapid, irreversible decay. We propose that the rate of inactivation of this mutant valyl-tRNA synthetase in vivo is a function of the ratio of deacylated to acylated tRNA(val) and that this ratio is a function of growth rate. The event which renders the valyl-tRNA synthetase susceptible to inactivation is likely to be the normal breakdown of the valyl-tRNA synthetase-valyl-adenylate complex during a cycle of aminoacylation of tRNA(val).

Acylation↗
PubMed2001

Recognition of tRNA by aminoacyl-tRNA synthetase from hyperthermophilic archaea, Aeropyrum pernix K1.

To study the recognition sites of tRNA for archaeal aminoacyl-tRNA synthetase, several aminoacyl-tRNA synthetase genes from hyperthermophilic archaeon, Aeropyrum pernix K1 were cloned and expressed. All the expressed enzymes showed extreme thermostability. Expressed threonyl-tRNA synthetase threonylated not only archaeal (A. pernix and Haloferax volcanii) threonine tRNAs but also Escherichia coli threonine tRNA. However, threonyl-tRNA synthetase from H. volcanii did not threonylate E. coli threonine tRNA.

Desulfurococcaceae↗
PubMed1993

Structural basis for transfer RNA aminoacylation by Escherichia coli glutaminyl-tRNA synthetase.

The structure of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNA2Gln and ATP refined at 2.5-A resolution reveals structural details of the catalytic center and allows description of the specific roles of individual amino acid residues in substrate binding and catalysis. The reactive moieties of the ATP and tRNA substrates are positioned within hydrogen-bonding distance of each other. Model-building has been used to position the glutamine substrate in an adjacent cavity with its reactive carboxylate adjacent to the alpha-phosphate of ATP; the interactions of the carboxyamide side chain suggest a structural rationale for the way in which the enzyme discriminates against glutamate. The binding site for a manganese ion has also been identified bridging the beta- and gamma-phosphates of the ATP. The well-known HIGH and KMSKS sequence motifs interact directly with each other as well as with the ATP, providing a structural rationale for their simultaneous conservation in all class I synthetases. The KMSKS loop adopts a well-ordered and catalytically productive conformation as a consequence of interactions made with the proximal beta-barrel domain. While there are no protein side chains near the reaction site that might function in acid-base catalysis, the side chains of two residues, His43 and Lys270, are positioned to assist in stabilizing the expected pentacovalent intermediate at the alpha-phosphate. Transfer of glutamine to the 3'-terminal tRNA ribose may well proceed by intramolecular catalysis involving proton abstraction by a phosphate oxygen atom of glutaminyl adenylate. Catalytic competence of the crystalline enzyme is directly shown by its ability to hydrolyze ATP and release pyrophosphate when crystals of the ternary complex are soaked in mother liquor containing glutamine.

Acylation↗
PubMed2006

The ribosome's response to codon-anticodon mismatches.

The ribosome is a molecular machine that synthesizes polypeptides from aminoacyl-tRNAs according to the sequence of the mRNA template. Codon reading by the anticodon of tRNA is controlled by a network of ribosome contacts that are specific for each position of the codon-anticodon duplex and involve A-minor RNA interactions. Rapid and accurate tRNA selection is accomplished by switching the conformation of the decoding site between accepting and rejecting mode, regardless of the thermodynamic stability of the respective codon-anticodon complexes or their interactions at the decoding site. The forward reactions are particularly sensitive to mismatches and determine the variations in the extent of misreading of near-cognate codons, both during initial selection and proofreading. This review emphasizes the progress made in understanding the mechanisms that determine recognition and selection of tRNA by the translational machinery.

Anticodon↗
PubMed1991

Structure and evolution of a group of related aminoacyl-tRNA synthetases.

A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate.

Adenosine Triphosphate↗
PubMed1983

The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor. Their purification, characterization, and the study of their interaction.

The glutamyl-tRNA synthetase from Bacillus subtilis has been purified to homogeneity. It is a monomer of Mr = 65,500 whose NH2-terminal sequence is Met-Asn-Glu-Val-Arg-Val-Arg-Tyr-Ser-Pro-Ser-Pro-Thr-Gly-His-Leu. The number of tryptic peptides indicates the absence of a significant amount of sequence duplication. Under certain conditions, this monomeric enzyme is co-purified with a polypeptide beta of Mr = 46,000, which increases the affinity of the enzyme about 10-fold for glutamate and for ATP, and stabilizes it against heat inactivation. gamma-Globulins prepared against the monomeric enzyme can inhibit completely the glutamyl-tRNA synthetase activity of a B. subtilis extract and precipitate from this extract both the monomeric enzyme and the regulatory factor beta. These anti-alpha immunoglobulins do nt precipitate pure beta. These results show that the glutamyl-tRNA synthetase of B. subtilis has a structure similar to that of the Escherichia coli enzyme (Lapointe, J., and Söll, D. (1972) J. Biol. Chem. 247, 4966-4974) and indicate that the beta factor has a function in the regulation of glutamyl-tRNA biosynthesis in vivo.

Amino Acid Sequence↗
PubMed1980

Transcription and processing of a yeast tRNA gene containing a modified intervening sequence.

The tRNA(3) (Leu) gene from yeast contains an intervening sequence of 32 nucleotides not present in mature tRNA. This sequence is transcribed and subsequently removed during the maturation of the RNA. To probe the involvement of this region of the gene in transcription and processing of the pre-tRNA(3) (Leu), the yeast DNA was cloned in plasmid pBR322 and a 21-base-pair DNA fragment corresponding to the lac operator was inserted into the intervening sequence. Insertion was done at a cleavage site for the restriction endonuclease Hpa I that occurs 19/20 base pairs from the 5' end of the intervening sequence. The parent and modified plasmids were then transcribed in a Xenopus germinal vesicle extract. RNA-fingerprint analysis of the transcription products revealed that both the tRNA(3) (Leu) gene and its modified counterpart were accurately transcribed. Transcription products corresponding to mature tRNA(3) (Leu) and pre-RNA(3) (Leu) with the normal and lac-containing intervening sequence were identified. Precursors extended at their 5' and 3' ends were also present. Both parent and modified genes were transcribed efficiently, and the various products accumulated in similar amounts, indicating that no deleterious effects on transcriptional competence, stability of the transcripts, or processing result from insertion of the 21-base-pair lac operator DNA. Incubation of pre-tRNA molecules that contained intervening sequences but were 5' and 3' mature with a yeast ribosomal wash fraction resulted in excision of the intervening sequence and, in the presence of ATP, ligation of the resulting half-tRNA molecules. The presence of RNA complementary to lac operator DNA neither inhibited the excision and splicing activities nor altered the site of the junction.

Base Sequence↗
PubMed2001

Yeast cytoplasmic and mitochondrial methionyl-tRNA synthetases: two structural frameworks for identical functions.

The yeast Saccharomyces cerevisiae possesses two methionyl-tRNA synthetases (MetRS), one in the cytoplasm and the other in mitochondria. The cytoplasmic MetRS has a zinc-finger motif of the type Cys-X(2)-Cys-X(9)-Cys-X(2)-Cys in an insertion domain that divides the nucleotide-binding fold into two halves, whereas no such motif is present in the mitochondrial MetRS. Here, we show that tightly bound zinc atom is present in the cytoplasmic MetRS but not in the mitochondrial MetRS. To test whether the presence of a zinc-binding site is required for cytoplasmic functions of MetRS, we constructed a yeast strain in which cytoplasmic MetRS gene was inactivated and the mitochondrial MetRS gene was expressed in the cytoplasm. Provided that methionine-accepting tRNA is overexpressed, this strain was viable, indicating that mitochondrial MetRS was able to aminoacylate tRNA(Met) in the cytoplasm. Site-directed mutagenesis demonstrated that the zinc domain was required for the stability and consequently for the activity of cytoplasmic MetRS. Mitochondrial MetRS, like cytoplasmic MetRS, supported homocysteine editing in vivo in the yeast cytoplasm. Both MetRSs catalyzed homocysteine editing and aminoacylation of coenzyme A in vitro. Thus, identical synthetic and editing functions can be carried out in different structural frameworks of cytoplasmic and mitochondrial MetRSs.

Acylation↗
PubMed1983

Loop stereochemistry and dynamics in transfer RNA.

The stereochemistry and the dynamics of two loops of yeast tRNA-asp, the thymine loop and the anticodon loop, are compared in the hope of a better understanding of the relationships between loop sequence and loop topology. Both loops are seven residues long and both present sharp turns after the second residue, U33 and psi 55, stabilized by hydrogen bonds between N3-H of the pyrimidine and the phosphates of C36 and A58 and stacking interactions of the pyrimidine ring with the phosphates of U35 and A57, respectively. In the thymine loop, the two purines following C56, A57 and A58, open up to leave space for the intercalation of the first invariant guanine residue of the D-loop, while the two pyrimidine bases, which follow A58, turn away from the stacking pattern of the thymine arm and stack instead with the last base pair of the dihydrouridine arm A15-U48. In the anticodon loop, however, the bases G34 to C38 form an helical stack in continuity with the anticodon stem on the 3'-end. At the same time C36 forms Watson-Crick hydrogen bonds with G34 of a twofold symmetrically related molecule. The anticodon-anticodon base pairing interactions between symmetrically-related molecules are stabilized by stacking with the modified base G37 on both sides of the triplet. Some comparisons are made with the structure of yeast tRNA-phe and some implications about the structure of mitochondrial tRNAs are discussed.

Anticodon↗
PubMed2000

Catalytic peptide of human glutaminyl-tRNA synthetase is essential for its assembly to the aminoacyl-tRNA synthetase complex.

Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.

Adenosine Triphosphate↗
PubMed1995

Protein-RNA recognition.

Specific interactions between RNAs and proteins are fundamental to many cellular processes, including the assembly and function of ribonucleoprotein particles (RNPs), such as ribosomes and spliceosomes and the post-transcriptional regulation of gene expression. Among the complexes studied to date are small RNAs bound to individual amino acids, tRNAs and tRNA fragments bound to their cognate aminoacyl-tRNA synthetases, and a variety of proteins bound to RNA single strands, hairpins, irregular helices, and tertiary structures stabilized by bound cations. Several proteins use a beta-sheet surface to bind RNAs, and others insert an alpha-helix into the widened major groove of a non-canonical RNA helix. Distortion or rearrangement of the RNA structure by bound protein is a common theme. The structural details of protein-RNA complexes are being resolved by nuclear magnetic resonance (NMR) and X-ray crystallography, but thorough thermodynamic analyses of recognition mechanisms have yet to be performed.

Amino Acid Sequence↗