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At least 667 records · Page 37Linked to original sources
PubMed1980

Fluorimetric study of the complex between yeast phenylalanyl-tRNA synthetase and tRNA-Phe. 2. Evidence for an asymmetric behaviour of the enzyme.

The variations of several spectroscopic properties of yeast tRNA-Phe and phenylalanyl-tRNA synthetase upon complex formation, were used to study the stoichiometry of the complex in different experimental conditions. In all cases, for the tRNA-Phe-enzyme complex, in the absence of other ligands, the saturations of the different conformational changes monitored for both macromolecules, are achieved at a 2:1 tRNA/enzyme stoichiometry. Phenylalanine does not modify this saturation. In contrast, the presence of 1 mM ATP induces an asymmetric behaviour of the synthetase: two tRNAs are still bound per enzyme molecule but the conformational change of the latter is completed upon binding of a single tRNA molecule.

Amino Acyl-tRNA Synthetases↗
PubMed1976

Phenylalanyl-tRNA synthetase from baker's yeast: specificity and quantitation of affinity elution with tRNA.

TRNAPhe is able to elute phenylalanyl-tRNA synthetase from cation exchangers in a 1:1 ratio. Elution of phenylalanyl-tRNA synthetase in a 1:1 ratio is also observed for four noncognate tRNAs investigated, specific for valine, serine, isoleucine and tyrosine. If protein mixtures are subjected to affinity elution the cognate pair [tRNAPhe-phenylalanyl-tRNA synthetase] is eluted first, followed by noncognate pairs. The unspecific elution is not influenced by complexation of phenylalanyl-tRNA synthetase with an analog of phenylalanyl-adenylate.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Structure of the yeast isoleucyl-tRNA synthetase gene (ILS1). DNA-sequence, amino-acid sequence of proteolytic peptides of the enzyme and comparison of the structure to those of other known aminoacyl-tRNA synthetases.

The ILS1 gene encoding for cytoplasmic isoleucyl-tRNA synthetase from Saccharomyces cerevisiae was subcloned from a 5.4-kb insert of the shuttle vector YEp13 to M13mp8 and M13mp9. Nucleotide sequence analysis of a 4.3-kb BamHI-HpaI fragment revealed a single open reading frame from which we deduced the amino-acid sequence of the enzyme. Independently obtained amino-acid sequence information from ten tryptic peptides of the purified enzyme confirmed the gene-derived structure. The enzyme is comprised of 1073 amino-acids consistent with earlier determinations of its molecular mass. The codon usage of ILS1 is typical of abundant yeast proteins. A significant homology to E. coli isoleucyl- and valyl-tRNA synthetases as well as to yeast valyl-tRNA synthetase was detected. The characteristic amino-acid residues of the aminoacyl-adenylate site and of the potential binding site of the 3'-end of tRNA found in other synthetases are present in the structure.

Amino Acid Sequence↗
PubMed1984

Recognition of tRNAs by aminoacyl-tRNA synthetases: Escherichia coli tRNAMet and E. coli methionyl-tRNA synthetase.

In previous work we identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (MetRS) (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. We have now investigated the aminoacylation activity of a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro by using T4 RNA ligase (EC 6.5.1.3). Base substitutions in the wobble position have been found to reduce aminoacylation rates by at least five orders of magnitude. Derivatives having base substitutions in the other two positions of the anticodon are aminoacylated 55-18,500 times slower than normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, which indicates that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of MetRS with functional groups on the nucleotide bases of the anticodon sequence. Several other aminoacyl-tRNA synthetases are known to require one or more anticodon bases for efficient aminoacylation of their tRNA substrates, and data from other laboratories suggest that anticodon sequences may be important for accurate discrimination between cognate and noncoagnate tRNAs by these enzymes.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Functional connectivity between tRNA binding domains in glutaminyl-tRNA synthetase.

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

Amino Acyl-tRNA Synthetases↗
PubMed2000

An intermediate step in the recognition of tRNA(Asp) by aspartyl-tRNA synthetase.

The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNA(Asp) from either T. thermophilus or Escherichia coli reveal a potential intermediate of the recognition process. The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes. While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains. The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 degrees C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state. Based on these data, a pathway for tRNA binding and recognition is proposed.

Anticodon↗
PubMed1995

Searching tRNA sequences for relatedness to aminoacyl-tRNA synthetase families.

tRNA sequences were analyzed for sequence features correlated with known classes of aminoacyl-tRNA synthetase enzymes. The tRNAs were searched for distinguishing nucleotides anywhere in their sequences. The analyses did not find nucleotides predictive of synthetase class membership. We conclude that such nucleotides never existed in tRNA sequences or that they existed and were lost from many of the tRNA sequences during evolution.

Amino Acid Sequence↗
PubMed1992

Footprinting evidence for close contacts of the yeast tRNA(Asp) anticodon region with aspartyl-tRNA synthetase.

Chemical footprinting experiments on brewer's yeast tRNA(Asp) complexed to its cognate aspartyl-tRNA synthetase are reported: they demonstrate that bases of the anticodon loop, including the anticodon itself, are in close proximity with the synthetase. Contacts were determined using dimethylsulfate as the probe for testing reactivity of guanine and cytosine residues in free and complexed tRNA. Results correlate with the decrease in aspartylation activity of yeast tRNA(Asp) molecules mutated at these contact positions and will be compared with other structural data arising from solution and crystallographic studies on the aspartic acid complex.

Alkylation↗
PubMed1984

Does the complex of aminoacyl-tRNA synthetases and tRNA-modifying enzymes prevent miscoding?

Several aminoacyl-tRNA synthetases of higher eukaryotes have always been found as multienzyme complexes. There are indications that these complexes can be associated with some tRNA-modifying enzymes. The function of such complexes is unclear. I have noticed that 6 out of 7 aminoacyl-tRNA synthetases most commonly occurring in complexes correspond to a group of tRNAs which must always contain a modified U in the first position of their anticodons. A hypothesis is proposed according to which association of 6 aminoacyl-tRNA synthetases with U-modifying enzymes can protect a cell from miscoding.

Amino Acids↗
PubMed1993

Random-splitting of tRNA transcripts as an approach for studying tRNA-protein interactions.

Location of phosphodiester bonds essential for aminoacylation of bovine tRNA(Trp) was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21, 22, 36-38, 57-59, 62 and 64 were critical for aminoacylation capacity of tRNA(Trp)-transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and in the regions presumably outside the contact area as well. These results indicate that for maintenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non-modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed in this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.

Acylation↗
PubMed1995

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

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

Anticodon↗
PubMed1991

Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase.

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).

Anticodon↗
PubMed1992

Crystallization of aspartyl-tRNA synthetase-tRNA(Asp) complex from Escherichia coli and first crystallographic results.

Crystals of the dimeric aspartyl-tRNA synthetase from Escherichia coli (molecular mass 132,000 Da) complexed with its cognate tRNA (molecular mass 25,000 Da) have been grown using ammonium sulfate as precipitant. The crystals belong to the orthorhombic space group C222(1) with unit cell parameters a = 102.75 A, b = 128.11 A, c = 231.70 A and diffract to 3 A. The asymmetric unit contains one monomer of the aspartyl-tRNA synthetase and one tRNA molecule.

Aspartate-tRNA Ligase↗
PubMed1981

Decreased protein synthesis by polysomes, tRNA and aminoacyl-tRNA synthetases isolated from senescent rat liver.

A cell-free in vitro protein-synthesizing system isolated from adult(10-13-month) and senescent(24-30-month) female Wistar rat liver is described. Optimal concentrations of polysomes, aminoacyl-tRNA synthetases, tRNA, Mg 2+, K+, and labeled amino acids are reported for this in vitro system. Both are rate and extent of protein synthesis are lower with the system from senescent liver compared to that from adult liver. Senescent polysomes, tRNA synthetases, and tRNA all contribute to this decrease. Free adult or senescent polysomes support protein synthesis to the same extent. However, compared to adult polysomes, senescent membrane-bound polysomes support less protein synthesis and account for an increased proportion of the total polysome fraction. Ribonuclease and protease activities are low in the adult and the senescent in vitro systems and do not account for the decreased protein synthesis with the latter.

Aging↗
PubMed1979

Improved separation of modified nucleosides from tRNA hydrolysates: the patterns of tRNA methylation in rat tissues.

A sensitive and reproducible method for the isolation of minor nucleosides derived from tRNA is described. The nucleosides obtained from enzymatic digestion of tRNA are separated into several groups using a QAE Sephadex column and increasing concentrations of boric acid in a step-wise manner. The nucleosides in each group are separated by isocratic elution from a preparative Partisil 10-SCX column and high-performance liquid chromatography at ambient temperature. With this method we have determined the patterns of tRNA methylation in vitro with extracts from rat bone, liver, kidney and adrenal glands. Although different tissues appear to contain the same tRNA methyltransferases, the patterns of methylated nucleosides are different.

Adrenal Glands↗
PubMed1990

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome.

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.

Anticodon↗
PubMed2001

Conserved amino acids near the carboxy terminus of bacterial tyrosyl-tRNA synthetase are involved in tRNA and Tyr-AMP binding.

Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino-terminal region, the active site domain, is more conserved (>36% identity). The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.

Adenosine Monophosphate↗
PubMed1996

A tyrosyl-tRNA synthetase recognizes a conserved tRNA-like structural motif in the group I intron catalytic core.

The Neurospora crassa mitochondrial (mt) tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns, in addition to aminoacylating tRNA(Tyr). Here, we compared the CYT-18 binding sites in the N. crassa mt LSU and ND1 introns with that in N. crassa mt tRNA(Tyr) by constructing three-dimensional models based on chemical modification and RNA footprinting data. Remarkably, superimposition of the CYT-18 binding sites in the model structures revealed an extended three-dimensional overlap between the tRNA and the group I intron catalytic core. Our results provide insight into how an RNA-splicing factor can evolve from a cellular RNA-binding protein. Further, the structural similarities between group I introns and tRNAs are consistent with an evolutionary relationship and suggest a general mechanism for the evolution of complex catalytic RNAs.

Base Sequence↗