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At least 631 records · Page 35Linked to original sources
PubMed1995

Crucial role of an idiosyncratic insertion in the Rossman fold of class 1 aminoacyl-tRNA synthetases: the case of methionyl-tRNA synthetase.

A few aminoacyl-tRNA synthetases are characterized by their ability to tightly bind a zinc atom. In the case of Escherichia coli methionyl-tRNA synthetase, a peptide of 21 residues (138--163) having a stable 3-D structure in solution is responsible for zinc binding [Fourmy, D., Meinnel, T., Mechulam, Y., & Blanquet, S. (1993) J. Mol. Biol. 231, 1066--1077; Fourmy, D., Dardel, F., & Blanquet, S. (1993) J. Mol. Biol. 231, 1078--1089]. This peptide, which belongs to a region connecting the two halves of the nucleotide-binding domain of methionyl-tRNA synthetase, is likely to form a modular domain close to the active center of the enzyme. In this study, two residues of the zinc-binding module, Asp138 and Arg139, are shown to contribute to the stabilization of the transition state of the reaction leading to the activation of methionine. Moreover, another residue, Phe135, located at the surface of the zinc-binding domain, is found to possibly guide the tRNA acceptor stem toward the active site of the enzyme during catalysis. The available data indicate an important functional role for the zinc-binding module of methionyl-tRNA synthetase, as well as for other modules connecting conserved secondary structure elements in the aminoacyl-tRNA synthetase family. The relation between the occurrence of such variable peptide modules and the expression of both substrate specificity and catalytic efficiency is discussed.

Adenosine Monophosphate↗
PubMed1998

The role of tryptophan residues in Escherichia coli arginyl-tRNA synthetase.

The effect of N-bromosuccinimide (NBS) on the activity of Escherichia coli arginyl-tRNA synthetase (ArgRS) was studied. The results showed that only one tryptophan residue was easy of access to the reagent and was closely related to enzyme activity. When all the five tryptophan residues in ArgRS were changed via site-directed mutagenesis singly into Ala, the aminoacylation activity of the Trp162 mutated enzyme decreased seriously, while the other four mutant enzymes retained almost the same activity as the native one. The oxidation of the five mutant enzymes with NBS demonstrated that only the mutation of Trp162 resulted in the loss of sensitivity to the reagent. These results strongly suggest that Trp162 is more accessible to NBS and is related to enzyme activity. Furthermore, the far-UV CD spectroscopy of the mutant enzyme ArgRS162WA showed little change in its secondary structure. Finally, studies on the kinetics of the mutant enzyme ArgRS162WA in aminoacylation reaction showed that the reduction in activity could be attributed to the decrease in the values of kcat and kcat/Km for arginine. The thermodynamic calculation indicates that this mutation causes a decrease of the binding energy by 2.7 kJ/mol. Our data suggest that Trp162 is involved in the binding of arginine and in the transition state stabilization.

Acylation↗
PubMed1996

Single sequence of a helix-loop peptide confers functional anticodon recognition on two tRNA synthetases.

The specific aminoacylation of RNA oligonucleotides whose sequences are based on the acceptor stems of tRNAs can be viewed as an operational RNA code for amino acids that may be related to the development of the genetic code. Many synthetases also have direct interactions with tRNA anticodon triplets and, in some cases, these interactions are thought to be essential for aminoacylation specificity. In these instances, an unresolved question is whether interactions with parts of the tRNA outside of the anticodon are sufficient for decoding genetic information. Escherichia coli isoleucyl- and methionyl-tRNA synthetases are closely related enzymes that interact with their respective anticodons. We used binary combinatorial mutagenesis of a 10 amino acid anticodon binding peptide in these two enzymes to identify composite sequences that would confer function to both enzymes despite their recognizing different anticodons. A single peptide was found that confers function to both enzymes in vivo and in vitro. Thus, even in enzymes where anticodon interactions are normally important for distinguishing one tRNA from another, these interactions can be 'neutralized' without losing specificity of amino-acylation. We suggest that acceptor helix interactions may play a role in providing the needed specificity.

Amino Acid Sequence↗
PubMed1978

Replacement of wybutine by hydrazines and its effect on the active conformation of yeast tRNAPhe.

The highly modified base wybutine (YWye) next to the anticodon of yeast tRNAPhe has been replaced by different hydrazine derivatives. The effect of the replacement on the activity of the tRNA has been studied in the heterologous aminoacylation with synthetase from E. coli and in the poly(U) directed binding to ribosomes from both yeast and E. coli. It was found that starting from tRNA-PheYWye the activity increased with increasing size, aromaticity, and stacking tendency of the substituent replacing YWye. It is concluded that YWye by the size of its aromatic system and by its stacking properties is particularly well suited for stabilizing the native conformation of tRNAPhe.

Escherichia coli↗
PubMed2004

Nuclear control of cloverleaf structure of human mitochondrial tRNA(Lys).

The evolutionary loss in eukaryotic cells of mitochondrial (mt) tRNA genes and of tRNA structural information in the surviving genes has led to the appearance of mt-tRNAs with highly unusual structural features. One such mt-tRNA is the human mt-tRNALys, which relies on post-transcriptional base modification to achieve correct three-dimensional structure. It has been shown that the in vitro transcript of human mt-tRNALys adopts a particular, non-cloverleaf structure when devoid of modified bases, while the native, fully modified tRNA shows the expected cloverleaf structure. Furthermore, a methyl group at position A9-N1, introduced chemically in an otherwise unmodified mt-tRNALys transcript, was found to induce a stable cloverleaf conformation, raising the question of how the specific methyltransferase recognizes the unmodified transcript. In order to shed light on this unusual case of tRNA maturation, the tRNA modification enzymes contained in protein extracts from either highly purified HeLa cell mitochondria or HeLa cell cytosol were first identified and compared, and then used to analyze the mt-tRNALys. An initial screening for modification activities, using as substrates unmodified in vitro transcripts of tRNA genes with well characterized structures, namely yeast cytosolic tRNAPhe, human cytosolic tRNA3Lys, and human mt-tRNAIle, revealed the presence of nine and 11 modification activities in the mitochondrial and cytosolic protein extracts, respectively, the mitochondrial extract including a tRNA (adenine-9,N1)-methyltransferase activity. The comparison of the level and kinetics of A9-N1 methylation and other secondary modifications in the unmodified, misfolded mt-tRNALys and in a cloverleaf-shaped structural mutant, engineered to adopt the tRNALys cloverleaf structure without post-transcriptional modifications, suggested strongly that the methylation of A9-N1 in tRNALys proceeds via a cloverleaf-shaped intermediate. Therefore, it is proposed that this intermediate is present in the in vitro transcript as part of a dynamic equilibrium, and that the mitochondrial protein extract contains an activity that stabilizes, by secondary modification, such a transient cloverleaf-shaped intermediate. Thus, countering the evolutionary loss of structural information in mt-tRNA genes, the mt-tRNA structure is maintained by a modification enzyme encoded in nuclear DNA.

Cytosol↗
PubMed1976

Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast.

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.

Amino Acyl-tRNA Synthetases↗
PubMed1994

Serine protease inhibitors block N-terminal arginylation of proteins by inhibiting the arginylation of tRNA in rat brains.

The tRNA mediated, posttranslational, N-terminal arginylation of proteins occurs in all eukaryotic cells. In nervous tissue, these reactions can be inhibited by endogenous molecules with a molecular weight of between one thousand and five thousand. In the present experiments, exogenous serine protease inhibitors (10(-5) M or less) but not other types of protease inhibitors, were found to be able to block the arginylation of protein in extracts of rat brain homogenates. Inhibition was not by the usual mode of action of protease inhibitors, but by interfering (non-competitively) with the charging of tRNA. Since arginylated proteins are rapidly ubiquitinated and degraded by cytosolic proteases, serine protease inhibitors may act to stabilize proteins by a dual mechanism of inhibiting arginylation as well as inhibiting serine proteases.

Animals↗
PubMed1993

Asparagine-135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket.

This work studies the structure-function relationships of Asn135, a residue situated in the GTP binding pocket of elongation factor Tu (EF-Tu). For this purpose we constructed EF-TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF-TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn135 side-chain does not recognize the exocyclic keto group of the guanine base. However, EF-TuN135D/D138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa-tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn135 is important for the correct folding of the nucleotide binding site and that EF-Tu.kirromycin can mediate the binding of aa-tRNA to the mRNA-programmed ribosomes independently of the native conformation of this site.

Asparagine↗
PubMed1998

Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp.

Ribonuclease P (RNase P) is an endonuclease that cleaves precursor tRNA to form the 5'-end of mature tRNA and is composed of a catalytic RNA subunit and a small protein subunit. The function of the protein component of Bacillus subtilis RNase P in catalysis of B. subtilis precursor tRNAAsp cleavage has been elucidated using steady-state kinetics, transient kinetics, and ligand affinity measurements to compare the functional properties of RNase P holoenzyme to RNase P RNA in 10 mM MgCl2, 100 mM NH4Cl. The protein component modestly affects several steps including </=10-fold increases in the rate constant for tRNA dissociation, the affinity of tRNA, and the rate constant for phosphodiester bond cleavage. However, the protein principally affects substrate binding, increasing the affinity of RNase P for pre-tRNAAsp by a factor of 10(4) as determined from both the ratio of the pre-tRNAAsp dissociation and association rate constants measured in 10 mM MgCl2 and a binding isotherm measured in 10 mM CaCl2 using gel filtration to separate enzyme-bound and free pre-tRNAAsp. Therefore, the main role of the protein component in RNase P is to facilitate recognition of pre-tRNA by enhancing the interaction between the enzyme and the 5'-precursor segment of the substrate, rather than stabilizing the tertiary structure of the folded RNA as has been observed for protein-facilitated group I intron self-splicing. Furthermore, the protein component maximizes the efficiency of RNase P under physiological conditions and minimizes product inhibition.

Bacillus subtilis↗
PubMedDate not supplied

[Properties of different classes of low molecular weight RNAs].

Low molecular weight (LMW) RNAs from early fish embryos were characterized by investigating their migration in polyacrylamide gels, solubility in 1.5 M NaCl, in vivo methylation by the methionine methyl group, metabolic stability, and effects of low doses of alpha-amanitin on their syntheses. At blastula stage, the mature tRNA and 4.3--4.8 S metabolically unstable molecules described as precursors of tRNA in pulse-chase experiments were found. The syntheses of stable methylated LMW RNAs, different from tRNA, were also shown. The sizes of these molecules, determined by their migration in polyacrylamide gels, were 5.3S; 5.8S; 6.3S and 6.9S These RNAs resemble the nuclear LMW RNAs, discovered in different animal cells, in infertilized eggs and developing embryos of sea urchin among them. The data obtained allow one to suppose an existance of a maturation stage during formation of these RNAs. The synthesis of a certain fraction of LMW RNAs (4.9S) was sensible to low doses of alpha-amanitin and therefore involved RNA-polymerase II.

Amanitins↗
PubMed1978

The effect of sulfhydryl reagents on the activity and stability of yeast ribosomes.

By titration with N-ethylmaleimide (MalNet) and p-chloromercuribenzoate (pCMB) of native yeast ribosomes, 40-43 SH groups were found, which represent about one third of the total SH groups present in these ribosomes. The reaction with pCMB proceeded fast, and that with MalNet was slow. The SH-blocking reaction led to inhibition of the ribosome activity. The activity of the MalNet-treated ribosomes in the polymerization reaction decreased concomitantly with the decrease of their activity in the enzymic and non-enzymic binding of phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA. pCMB completely inactivated the ribosomes which seems to indicate that this reagent modified some SH groups which were not accessible to MalNet. The ribosome stability was affected by the blocking of the SH groups; in the buffered 0.5 M-KCl solution a significant portion of protein was detached from the modified ribosomes. Thus it appears that the SH groups of ribosomal proteins play some role in maintaining the ribosome structure.

Chloromercuribenzoates↗
PubMed1991

Selection of suppressor methionyl-tRNA synthetases: mapping the tRNA anticodon binding site.

Accurate aminoacylation of a tRNA by Escherichia coli methionyl-tRNA synthetase (MTS) is specified by the CAU anticodon. A genetic screening procedure was designed to isolate MTS mutants able to aminoacylate a methionine amber tRNA (CUA anticodon). Selected suppressor MTS enzymes all possess one or several mutations in the vicinity of Trp-461, a residue that is the major contributor to the stability of complexes formed with tRNAs having the cognate CAU anticodon. Analysis of catalytic properties of purified suppressor enzymes shows that they have acquired an additional specificity toward the amber anticodon without complete disruption of the methionine anticodon site. It is concluded that both positive and negative discrimination toward the binding of tRNA anticodon sequences is restricted to a limited region of the synthetase, residues 451-467.

Anticodon↗
PubMed1999

Characterization of zinc-depleted alanyl-tRNA synthetase from Escherichia coli: role of zinc.

To evaluate the role of zinc in Escherichia coli alanyl-tRNA synthetase, hydrodynamic measurements and circular dichroism spectra were obtained for the zinc-depleted protein and compared with those of the native enzyme. At a protein concentration of 5 mg ml(-1), pH 7.5, the sedimentation coefficient (s(20,w)) was 6.3 S and was virtually independent of temperature between 10 and 37 degrees C, similar to the results reported for the native form. However, the s(20,w) now decreased significantly as the concentration increased, indicative of a possible change in conformation. The s(20,w) value did not appear to change as the pH was increased to 9.5. In standard buffer with 3.3 M added urea, a single peak with a s(20,w) of 3.6 S was obtained and with 6.6 M added urea, a peak with a s(20,w) of 2.7 S was seen. Added Gd-HCl (6 M) gave a single peak with s(20,w) of 2. 0 S. Like the native form, laser light scattering studies indicated some heterogeneity and a radius of 6.4 nm which was virtually independent of concentration and temperature in the range of 10-37 degrees C. At 25 degrees C, a diffusion coefficient (D(20,w)) of 3.3 x 10(-7) cm(2) s(-1) was obtained. The combination of s(0)(20,w) and D(20,w) yielded a molecular mass of approximately 179 kDa, which is slightly less than that reported for the native dimeric form (186 kDa). The intrinsic viscosity at 25 degrees C was extrapolated to 5. 3 ml g(-1), a value significantly higher than that reported for the native form, which increased with temperature. These results indicate some conformational and flexibility changes from the native to the zinc-depleted form, which may explain differences in activity. Furthermore, urea denaturation experiments demonstrate the role of zinc in stabilization of AlaRS structure.

Alanine-tRNA Ligase↗
PubMed1979

Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli.

Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.

Amino Acyl-tRNA Synthetases↗