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At least 865 records · Page 48Linked to original sources
PubMed2006

A previously unidentified activity of yeast and mouse RNA:pseudouridine synthases 1 (Pus1p) on tRNAs.

Mouse pseudouridine synthase 1 (mPus1p) was the first vertebrate RNA:pseudouridine synthase that was cloned and characterized biochemically. The mPus1p was previously found to catalyze Psi formation at positions 27, 28, 34, and 36 in in vitro produced yeast and human tRNAs. On the other hand, the homologous Saccharomyces cerevisiae scPus1p protein was shown to modify seven uridine residues in tRNAs (26, 27, 28, 34, 36, 65, and 67) and U44 in U2 snRNA. In this work, we expressed mPus1p in yeast cells lacking scPus1p and studied modification of U2 snRNA and several yeast tRNAs. Our data showed that, in these in vivo conditions, the mouse enzyme efficiently modifies yeast U2 snRNA at position 44 and tRNAs at positions 27, 28, 34, and 36. However, a tRNA:Psi26-synthase activity of mPus1p was not observed. Furthermore, we found that both scPus1p and mPus1p, in vivo and in vitro, have a previously unidentified activity at position 1 in cytoplasmic tRNAArg(ACG). This modification can take place in mature tRNA, as well as in pre-tRNAs with 5' and/or 3' extensions. Thus, we identified the protein carrying one of the last missing yeast tRNA:Psi synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.

Animals↗
PubMed1983

Function and fidelity of aging tRNA: in vivo acylation, analog discrimination, synthetase binding, and in vitro translation.

The liver transfer RNAs for valine and lysine were completely acylated in vivo, as judged by periodate oxidation, at 4 and 24 months of age in male Sprague-Dawley rats. In vitro acylation capacity for whole tRNA populations from rat livers is decreased, but this is evidently not deleterious in vivo. Several halogenated phenylalanines were synthesized and their effects upon acylation capacity for phenylalanine were examined. Synthetases bound to young (3 month) and old (24 month) tRNAs discriminated differently between p-chlorophenylalanine and authentic phenylalanine; synthetase with young tRNA was less able to discriminate than with old tRNA. Purified tRNAphe from old rats did not form ultraviolet-induced crosslinks to purified phenylalanyl tRNA synthetase as well as young tRNAphe. In vitro translation of encephalomyocarditis virus, hemoglobin, and ovalbumin mRNAs was effective, using tRNAs of young or old Sprague-Dawley or Fischer 344 rat livers, although, when the old tRNA was supplied, the product synthesized per unit tRNA was reduced. All of the protein products were synthesized with all tRNAs, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We conclude that tRNA is capable of normal functions in livers of aging rats, is probably modification deficient, and is unlikely to produce protein errors.

Acylation↗
PubMed2007

Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine.

An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins.

Amino Acid Sequence↗
PubMed1984

Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase.

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl-tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and T psi arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable T psi stem. When alkylation by ethylnitrosourea was studied with the tRNATrp X tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase X tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.

Alkylation↗
PubMed1998

Aminoacylation of hypomodified tRNAGlu in vivo.

The highly specific interaction of each aminoacyl-tRNA synthetase and its substrate tRNAs constitutes an intriguing problem in protein-RNA recognition. All tRNAs have the same overall three-dimensional structure in order to fit interchangeably into the translational apparatus. Thus, the recognition by aminoacyl-tRNA synthetase must be more or less limited to discrimination between bases at specific positions within the tRNA. The hypermodified nucleotide 5-methylaminomethyl-2-thiouridine (mnm5s2U) present at the wobble position of bacterial tRNAs specific for glutamic acid, lysine and possibly glutamine has been shown to be important in the recognition of these tRNAs by their synthetases in vitro. Here, we have determined the aminoacylation level in vivo of tRNAGlu, tRNALys, and tRNA1GIn in Escherichia coli strains containing undermodified derivatives of mnm5s2U34. Lack of the 5-methylaminomethyl group did not reduce charging levels for any of the three tRNAs. Lack of the s2U34 modification caused a 40% reduction in the charging level of tRNAGlu. Charging of tRNALys and tRNA1Gln were less affected. There was no compensating regulation of expression of glutamyl-tRNA synthetase because the relative synthesis rate was the same in the wild-type and mutant strains. These results indicate that the mnm5U34 modification is not an important recognition element in vivo for the glutamyl-tRNA synthetase. In contrast, lack of the s2U34 modification reduced the efficiency of charging by at least 40%. This is the minimal estimate because the turn-over rate of Glu-tRNAGlu was also reduced in the absence of the 2-thio group. Lack of either modification did not affect mischarging or mistranslation.

Base Sequence↗
PubMed1979

A synthetic tyrosine suppressor tRNA gene with an altered promoter sequence. Its cloning and relative expression in vivo.

The total synthesis of a tyrosine suppressor tRNA gene with a modified promoter is described. The alteration involves the replacement of the four G:C base pairs immediately preceding the start point of transcription by A:T base pairs. The new sequence contains the recognition sequence for the HindIII restriction endonuclease at the transcriptional start point, thus permitting fusion of the structural gene with promoters containing independent sequence modifications. The construction, cloning, and biological activity of several recombinant DNAs containing the tRNA gene with the modified promoter are described. The expression of this gene in vivo is compared with that of both the unmodified synthetic suppressor gene and a naturally occurring tyr su3+ gene cloned onto a multicopy plasmid.

Base Sequence↗
PubMed1993

Influence of tRNA tertiary structure and stability on aminoacylation by yeast aspartyl-tRNA synthetase.

Mutations have been designed that disrupt the tertiary structure of yeast tRNA(Asp). The effects of these mutations on both tRNA structure and specific aspartylation by yeast aspartyl-tRNA synthetase were assayed. Mutations that disrupt tertiary interactions involving the D-stem or D-loop result in destabilization of the base-pairing in the D-stem, as monitored by nuclease digestion and chemical modification studies. These mutations also decrease the specificity constant (kcat/Km) for aspartylation by aspartyl-tRNA synthetase up to 10(3)-10(4) fold. The size of the T-loop also influences tRNA(Asp) structure and function; change of its T-loop to a tetraloop (-UUCG-) sequence results in a denatured D-stem and an almost 10(4) fold decrease of kcat/Km for aspartylation. The negative effects of these mutations on aspartylation activity are significantly alleviated by additional mutations that stabilize the D-stem. These results indicate that a critical role of tertiary structure in tRNA(Asp) for aspartylation is the maintenance of a base-paired D-stem.

Acylation↗
PubMed1979

Isolation of a single polypeptide leucyl-tRNA synthetase from bakers' yeast.

A single polypeptide of leucyl-tRNA synthetase (LRS) has been purified from budding bakers' yeast by a modification of the procedure published earlier. On denaturing polyacrylamide gel electrophoresis LRS was one band corresponding to molecular weight of 120,000 +/- 5,000 daltons. Variable amounts of LRS with a similar molecular weight but which dissociated into equal subunits of 58,000 were also isolated. The affinities (KM) for substrates for this form of the enzyme were similar to those previously reported for the dimeric form of the enzyme.

Amino Acyl-tRNA Synthetases↗
PubMed1981

Fluorimetric study of yeast tRNAPheCCF in the complex with phenylalanyl-tRNA synthetase. Evidence for a correlation between the structural adaptation of both macromolecules and the appearance of the acylation activity.

The fluorescence properties of yeast tRNAPheCCF (tRNAPhe in which the 3'-terminal adenosine has been replaced by formycin) and tRNAPheCCFoxi-red (tRNAPheCCF after periodate oxidation followed by borohydride reduction) were studied in the complex with the cognate aminoacyl-tRNA synthetase. In both cases a conformational change affecting the 3' end was observed in a magnesium concentration range close to 1 mM. The modification of formycin fluorescence could be ascribed simultaneously to the existence of a tautomeric equilibrium of the fluorescent probe and to a pH effect raising from a prototropic effect at the active site of phenylalanyl-tRNA synthetase, and to a partial destacking of the 3'-formycin from the adjacent C residue. The observed transconformation, which can be related to the structure modification of the anticodon loop previously reported [Ehrlich, Lefèvre, and Remy (1980) Eur. J. Biochem. 103, 145-153], takes place in the magnesium concentration range allowing the transfer of the activated amino acid from the adenylate to the tRNA. The interconnection between the anticodon loop and the accepting end was further supported by the observation that wybutine excision hinders the specific structure modification of 3'-formycin upon binding to the synthetase. The tRNAPhe transconformations occurring in the complex with the cognate synthetase probably reflect a reciprocal adaptation of both macromolecules which might lead to the optimal aminoacylation velocity and thus contribute to the specificity of aminoacylation, since it was previously established that this specificity relies more strongly on the kinetics of the reaction than on a discrimination of tRNAs according to different affinities.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Primary structure of bovine liver tRNATrp.

Purified tRNATrp from bovine liver, accepting 1700 pmol tryptophan per A260nm unit, was completely digested with pancreatic ribonuclease and T1 ribonuclease. The sequences of the resulting oligonucleotides were determined and the primary structure of the tRNA was deduced. These analyses showed numerous incomplete post-transcriptional modifications, and several positions heterogenously occupied by two different nucleotides, which lead us to think that in bovine liver there exist a mixture of several tRNATrp.

Animals↗
PubMed1992

Codon-anticodon pairing. A model for interacting codon-anticodon duplexes located at the ribosomal A- and P-sites.

The interaction between two codon-anticodon duplexes of the ribosomal A- and P-site-bound tRNAs is the key feature of the proposed model. This interaction prohibits non-canonical base pairing at the first and second positions of the codon and controls base pairing at the third position (wobbling rules ensuing from the model are in good accord with those generated from experiments). The model is capable of predicting codon context effects. It follows from the model that modifications of the first anticodon residue of the P-site tRNA can affect the stability of the A-site duplex, and that the translation of a DNA single chain analogue of mRNA should be accompanied by non-canonical base pairing at all three positions of the codon. These predictions of the model can be subjected to experimental tests.

Anticodon↗
PubMed1995

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV-1 reverse transcriptase: tRNA(Lys,3) activation.

HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.

DNA↗
PubMed2003

RNA-modifying enzymes.

A bewildering number of post-transcriptional modifications are introduced into cellular RNAs by enzymes that are often conserved among archaea, bacteria and eukaryotes. The modifications range from those with well-understood functions, such as tRNA aminoacylation, to widespread but more mysterious ones, such as pseudouridylation. Recent structure determinations have included two types of RNA nucleobase modifying enzyme: pseudouridine synthases and tRNA guanine transglycosylases.

Binding Sites↗
PubMed1996

Strategies for achieving high-level expression of genes in Escherichia coli.

Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli.

Biotechnology↗
PubMed1995

Relaxing the substrate specificity of an aminoacyl-tRNA synthetase allows in vitro and in vivo synthesis of proteins containing unnatural amino acids.

It has previously been demonstrated that the unnatural amino acid p-Cl-phenylalanine can be attached to tRNA(Phe) by a modified phenylalanyl-tRNA synthetase with relaxed amino acid substrate specificity. We show that this modification to the translational machinery of Escherichia coli is the only requirement for the incorporation of either p-Cl- or p-Br-phenylalanine into full-length luciferase in vitro. The incorporation of p-Cl-phenylalanine was also demonstrated in vivo using a suitably modified host strain. These results represent the first description of the incorporation into a protein in vivo of an unnatural amino acid which is normally rejected by the cellular translational machinery.

Chaperonins↗
PubMed1982

A comparative study of essential arginine residues in Gramicidin S synthetase 2 and isoleucyl tRNA synthetase.

In gramicidin S synthetase 2 (GS 2) from Bacillus brevis, L-proline, L-valine, L-ornithine, and L-leucine activations to aminoacyl adenylates are progressively inhibited by phenylglyoxal. The inactivation of GS 2 obeys pseudo-first-order kinetics. ATP completely prevents inactivation of GS 2 by phenylglyoxal, whereas amino acids only partially prevent it. In the presence of ATP, four arginine residues per mol of GS 2 are protected from modification by phenylglyoxal as determined by amino acid analysis and the incorporation of [7-14C]phenylgloxal into the enzyme protein, indicating that a single arginine residue is necessary for each amino acid activation. In isoleucyl tRNA synthetase from Escherichia coli, phenylglyoxal inhibits activation of L-isoleucine to isoleucyl adenylate. ATP completely prevents inactivation, although isoleucine only partially prevents it. One arginine residue of isoleucyl tRNA synthetase is protected by ATP from modification by phenylglyoxal, suggesting that a single arginine residue is essential for isoleucine activation. These results support the involvement of arginine residues in ATP binding with GS 2 or isoleucyl tRNA synthetase, and thus indicate that arginine residues of amino acid activating enzymes are essential for the formation of aminoacyl adenylates in both nonribosomal and ribosomal peptide biosynthesis.

Adenosine Triphosphate↗
PubMed2005

A new mechanism for mtDNA pathogenesis: impairment of post-transcriptional maturation leads to severe depletion of mitochondrial tRNASer(UCN) caused by T7512C and G7497A point mutations.

We have studied the consequences of two homoplasmic, pathogenic point mutations (T7512C and G7497A) in the tRNA(Ser(UCN)) gene of mitochondrial (mt) DNA using osteosarcoma cybrids. We identified a severe reduction of tRNA(Ser(UCN)) to levels below 10% of controls for both mutations, resulting in a 40% reduction in mitochondrial protein synthesis rate and in a respiratory chain deficiency resembling that in the patients muscle. Aminoacylation was apparently unaffected. On non-denaturating northern blots we detected an altered electrophoretic mobility for G7497A containing tRNA molecules suggesting a structural impact of this mutation, which was confirmed by structural probing. By comparing in vitro transcribed molecules with native RNA in such gels, we also identified tRNA(Ser(UCN)) being present in two isoforms in vivo, probably corresponding to the nascent, unmodified transcripts co-migrating with the in vitro transcripts and a second, faster moving isoform corresponding to the mature tRNA. In cybrids containing either mutations the unmodified isoforms were severely reduced. We hypothesize that both mutations lead to an impairment of post-transcriptional modification processes, ultimately leading to a preponderance of degradation by nucleases over maturation by modifying enzymes, resulting in severely reduced tRNA(Ser(UCN)) steady state levels. We infer that an increased degradation rate, caused by disturbance of tRNA maturation and, in the case of the G7497A mutant, alteration of tRNA structure, is a new pathogenic mechanism of mt tRNA point mutations.

Aminoacylation↗
PubMed1990

Heterogeneities in vertebrate tRNAs(Trp) avian retroviruses package only as a primer the tRNA(Trp) lacking modified m2G in position 7.

Bovine, rabbit and chicken tRNA(Trp) species and tRNA(Trp) packaged in avian myeloblastosis virus were separated and purified using two-dimensional gel electrophoresis and their primary structures were determined. Two major tRNA(Trp) species (1 and 2) were identified in beef and rabbit, two minor ones (3 and 4) in beef and only one minor in rabbit. Their structures differ by 4 nucleotide substitutions located in the D, S and T loops (positions 16, 47, 57 and 59). Species 3 and 4 differ from one another by only one nucleotide at position 2. Differences between tRNA(Trp) species were also observed in the extent of methylation of some nucleotides. Chicken tRNA(Trp) presents only one species similar to the mammalian type 1 tRNA(Trp). In the case of the three studies animals this tRNA could be separated into two subspecies, which differ by a post-transcriptional modification of nucleotide 7 in the acceptor stem: G or m2G. However only the nonmethylated species is used as the primer of DNA-RNA directed retrotranscription since it is only that form which was found in avian retroviruses. The methylation of G to m2G at position 7 could thus prevent the recognition of tRNA(Trp) by retroviral protein(s) responsible for the selective packaging of the primer tRNA(Trp).

Animals↗