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At least 163 records · Page 9Linked to original sources
PubMed1990

Growth rate dependence of transfer RNA abundance in Escherichia coli.

We have tested the predictions of a model that accounts for the codon preferences of bacteria in terms of a growth maximization strategy. According to this model the tRNA species cognate to minor and major codons should be regulated differently under different growth conditions: the isoacceptors cognate to major codons should increase at fast growth rates while those cognate to minor codons should decrease at fast growth rates. We have used a quantitative Northern blotting technique to measure the abundance of the methionine and the leucine isoacceptor families over growth rates ranging from 0.5 to 2.1 doublings per hour. Five tRNA species that are cognate to major codons (tRNA(eMet), tRNA(1fMet), tRNA(2fMet), tRNA(1Leu) and tRNA(3Leu) increase both as a relative fraction of total tRNA and in absolute concentration with increasing growth rates. Three tRNA species that are cognate to minor codons (tRNA(2Leu), tRNA(4Leu) and tRNA(5Leu) decrease as a relative fraction of total RNA and in absolute concentration with increasing growth rates. These data suggest that the abundances of groups of tRNA species are regulated in different ways, and that they are not regulated simply according to isoacceptor specificity. In particular, the data support the growth optimization model for codon bias.

Base Sequence↗
PubMed1997

Transfer RNA docking pair model in the ribosomal pre- and post-translocational states.

A consensus has been reached that the conformation of the anticodon-codon interactions of two adjacent tRNA molecules on the ribosome is a Sundaralingam-type (S-type). Even if it is kept to the S-type, there are still various possibilities. Various experimental data have been supporting an idea that the conformation of A-site tRNA is different from that of P-site tRNA. Those data as well as the recent result of Brimacombe and co-workers that U20:1 of lupin tRNAmMetbound to the A-site was cross-linked to a region, 875-905, of 23S rRNA in combination with the other recent findings of Nierhaus and co-workers about the spin-contrast method of neutron diffraction of the ribosome and the better accessible nucleotide patterns of phosphorothioated tRNAs on the ribosome have led to a new tRNA docking pair model, in which the highly conserved G18 and G19 of D-loop in A-site tRNA and C56 and C61 of TpsiC-loop in P-site tRNA base pair along with the conventional base pairs of adjacent codon-anticodon interactions. This A-P tRNA pair model can be translocated to the P-E tRNA pair model without changing the conformation except the ACCA termini, keeping the position of the growing nascent polypeptide chain.

Base Composition↗
PubMed1983

Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA.

The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNAMetf has been further characterized. This substituent is responsible for the 19F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution 19F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA. Similar highfield 19F signals have now been observed in the spectra of two other purified fluorinated E. coli tRNAs, tRNAMetm and tRNAVal1, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent. Comparison with 19F spectrum of the model compound 5'-deoxy-5-fluoro-5,6-dihydrouridine (dH56FUrd) (delta FUra = -31.4 ppm; JHF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring. dH56FUrd is considerably more alkali labile than 5,6-dihydrouridine (H56Urd). At pH 8.9, where H56Urd is stable, dH56FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a 19F resonance at - 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA. The 19F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the 19F signal at - 15 ppm. Hydrolysis of this tRNA with RNAase T2 produces a sharp doublet 33 ppm upfield (JHF = 45 Hz). Similarities of the 19F chemical shift and coupling constant to those of dH56FUrd, allows assignment of the peak at -33 ppm to an intact fluorodihydrouridine residue in the tRNA. Our results demonstrate that FUra residues incorporated into E. coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine. This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA.

Escherichia coli↗
PubMed1983

Proton exchange rates in transfer RNA as a function of spermidine and magnesium.

Solvent exchange rates of selected protons were measured by NMR saturation recovery for E. coli tRNAVal, E. colifMet and yeast tRNAPhe, at temperatures from 20 to 40 degrees C, in the presence of 0.12M Na+ and various levels of added spermidine. tRNAVal was also studied with added Mg++. The exchange rates in zero spermidine and Mg++ indicate early melting of the U8 A14 interaction, in accord with thermodynamic melting studies. Exchange rates for secondary protons suggest early melting of the T stem in tRNAfMet and the acceptor stem in tRNAPhe, in contradiction with melting transition assignments from thermodynamic work. Addition of 10 spermidines per tRNA stabilizes the secondary and tertiary interactions more effectively than added Na+, but less so than Mg++. Added spermidine has the curious effect of increasing the exchange rate of the psi 55 N1 proton, while protecting the psi 55 N3 proton from exchange in all three tRNA's. Added Mg++ has the same effect on tRNAVal.

Escherichia coli↗
PubMed1980

Transfer RNA labeling of Escherichia coli methionyl-tRNA transformylase.

Homogeneous methionyl-tRNA transformylase from Escherichia coli can react with periodate-treated tRNAMetf to form a Schiff's base through a free amino group (probably the epsilon-amino group of a lysine) and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. This reaction is reflected by the loss of activity of the enzyme at saturating tRNA dialdehyde and upon reduction of the Schiff's base by NaBH4. the kinetics of inactivation (37 degrees C, pH 8.5) level off at 50% of the initial enzymic activity. In the presence of 2 mM cyanohydridoborate, a mild reducing agent which leaves intact the reacting aldehyde groups of oxidized tRNA but continuously reduces the Schiff's base in equilibrium, the activity of the enzyme can be destroyed by 100%, at a rate of 0.044 min-1, with the parallel covalent incorporation of close to one tRNA molecule per enzyme molecule. Selectivity of the labeling is also supported by the demonstration that, prior to Schiff's base formation, modified tRNA binds the transformylase with equilibrium constant and stoichiometry in good agreement with those for the active binding of unmodified tRNa. Moreover intact tRNA competes for the inactivation by the dialdehyde derivative with an affinity constant identical to that for its active binding to the enzyme

Acyltransferases↗
PubMed1994

Energy cost of translational proofreading in vivo. The aminoacylation of transfer RNA in Escherichia coli.

In many cases, the intrinsic binding energies of amino acids to aminoacyl-tRNA synthetases are inadequate to give the required accuracy of translation. This has necessitated the evolution of a second determinant of specificity, proofreading, or editing mechanisms that involve the expenditure of energy to remove errors. Studies of an error-editing function of bacterial methionyl-tRNA synthetase have led to the discovery of a distinct chemical mechanism of editing and to molecular dissection of the dual synthetic-editing function of the active site of the synthetase. Studies have also established the importance of proofreading in living cells and allowed direct measurements of energy costs associated with editing in vivo. An unexpected outcome of these studies was a discovery of functional and structural similarities between methionyl-tRNA synthetase and S-adenosylmethionine synthetase, suggesting an evolutionary relationship between the two proteins. The mechanism of editing involves a nucleophilic attack of a sulfur atom on the side chain of homocysteine in homocysteinyl adenylate on its carbonyl carbon, yielding homocysteine thiolactone. The model of the active site of methionyl-tRNA synthetase derived from structure-function studies explains how the active site partitions amino acids between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions of active site residues Trp305 and Tyr15 with the side chain of methionine prevent the cognate amino acid from entering the editing pathway. These interactions are missing in the case of the smaller side chain of the noncognate homocysteine, which therefore enters the editing pathway. Homocysteine thiolactone is formed as a result of editing of homocysteine by methionyl-tRNA synthetase in bacteria, yeast, and some cultured mammalian cells. In mammalian cells, enhanced synthesis of homocysteine thiolactone, is, thus far, associated with oncogenic transformation. In E. coli, most of the energy cost of proofreading by methionyl-tRNA synthetase is due to editing of the incorrect product, homocysteinyl adenylate.

Adenosine Triphosphate↗
PubMed1987

Autoreactive epitope defined as the anticodon region of alanine transfer RNA.

Autoantibodies to aminoacyl-transfer RNA (tRNA) synthetases are common in the human autoimmune diseases polymyositis and dermatomyositis. Sera of the PL-12 specificity contain separate antibodies reacting with alanyl-tRNA synthetase and alanine tRNA (tRNAAla). The antibodies to tRNA recognize at least six distinguishable human tRNAAla species grouped into two sequence families. The antibody-reactive determinants on the tRNA were identified through ribonuclease protection and oligonucleotide binding experiments. The antibody binding site is a seven- to nine-nucleotide sequence containing the anticodon loop and requires an intact anticodon. No requirement for anticodon stem structure or sequence is observed, although the 5' portion of the stem is protected from nuclease attack. Antibodies from several patients appear to share the same specificitym, indicating that the antibodies are induced by a unique sequence feature in the immunogen.

Alanine-tRNA Ligase↗
PubMed1999

Transfer RNA genes and their significance to codon usage in the Pseudomonas aeruginosa lamboid bacteriophage D3.

Using tRNAscan-SE and FAStRNA we have identified four tRNA genes in the delayed early region of the bacteriophage D3 genome (GenBank accession No. AF077308). These are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA). The D3 Thr- and Gly-tRNAs recognize codons, which are rarely used in Pseudomonas aeruginosa and presumably, influence the rate of translation of phage proteins. BLASTN searches revealed that the D3 tRNA genes have homology to tRNA genes from Gram-positive bacteria. Analysis of codon usage in the 91 ORFs discovered in D3 indicates patterns of codon usage reminiscent of Escherichia coli or P. aeruginosa.

Bacteriophages↗
PubMed1987

19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA.

The use of 19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 (anticodon FAC). 19F n.m.r. spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A,G,C,U), which contain the valine codon GpUpA. Structural changes in the tRNA were monitored via the 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule. Binding of GpUpApA, which is complementary to the anticodon and the 5'-adjacent FUra 33, shifts two resonances in the 19F spectrum. One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon. The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the 19F spectrum of 19F-labeled tRNA(Val)1. Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon. The association constant for the binding of GpUpApA to fluorinated tRNA(Val)1, obtained by Scatchard analysis of the n.m.r. results, is in good agreement with values obtained by other methods. On the basis of these results, we assign peak F in the 19F n.m.r. spectrum of 19F-labeled tRNA(Val)1 to FUra 33. This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the 19F spectrum of fluorinated tRNA(Val)1 are specifically decreased after partial hydrolysis with nucleass S1 under conditions leading to cleavage in the anticodon loop. The downfield shift of peak F occurs only with adenosine in the 3'-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H. The possibility is discussed that this base-specific interaction between the 3'-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5'-stacked conformation of the anticodon loop. Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.

Anticodon↗
PubMed1999

Transfer RNA biogenesis: A visa to leave the nucleus.

Only correctly folded and mature tRNAs can leave the nucleus and enter the cytoplasm. Surprisingly, tRNA-aminoacylation has been found to occur, not only in the cytosol, but also inside the nucleus, where it may act as an additional proofreading step and facilitate the export of 'ready-to-function' aminoacyl-tRNAs.

Animals↗
PubMed1989

Queuine is incorporated into brain transfer RNA.

Pig brain tRNA was assayed for the presence of queuosine in the first position of the anticodon for each of the Q-family of tRNAs (aspartyl, asparaginyl, histidyl and tyrosyl). The brain tRNA was aminoacylated with each of the four amino acids and the aminoacylated tRNA's analyzed by RPC-5 chromatography. The results of this study show that for all four tRNAs of the family, queuine is substituted for guanine in virtually 100% of the anticodons. Therefore, it can be concluded that queuine is able to cross the blood-brain barrier and that brain contains guanine-queuine tRNA transglycosylase, the enzyme responsible for the excision of guanine from the original transcripts of these tRNAs and insertion of queuine. The determination of whether the tRNA contained queuine was made from the elution profile of the RPC-5 chromatograms and the results confirmed by a change in the RPC-5 elution profile when the tRNAs were reacted with BrCN or NaIO4.

Animals↗
PubMed1979

Transfer RNA control of the activation of isomeric tRNATrp's.

Previous studies of the homologous aminoacylations of Escherichia coli and yeast tRNATrp's terminating in 2'- and 3'-deoxyadenosine established that E. coli tryptophanyl-tRNA synthetase activates its cognate tRNA preferentially on the 2' position, while the corresponding yeast enzyme utilizes the 3' position on its homologous substrate tRNA. As this seemed to be the only change in positional specificity during evolution, the heterologous activations were investigated in an effort to determine the basis for this change. Remarkably, E. coli tRNATrp terminating in 3'-deoxyadenosine was found to be the preferred substrate for both the E. coli and yeast activating enzymes, while the same tryptophanyl-tRNA synthetase preparations both activated the isomeric yeast tRNATrp's preferentially on the 3' position. Thus, the preferred position of activation was found to be specified by the tRNA rather than the activating enzyme and, additionally, to be due to some process not reflected in initial velocity measurements. The variable utilization of individual modified aminoacyl-tRNA's as substrates in an enzyme-catalyzed deacylation process appears to provide the most likely explanation for the experimental observations.

Amino Acyl-tRNA Synthetases↗
PubMed2005

RNA transfer by electroporation into mature dendritic cells leading to reactivation of effector-memory cytotoxic T lymphocytes: a quantitative analysis.

Previous studies have analyzed transfer of RNA-encoded tumor-associated antigens (TAAs) into immature dendritic cells (DCs) because of their exceptional ability to internalize antigens. Concerns have been raised regarding the use of immature DCs in clinical studies because of their capacity to tolerize T cells. Therefore, we focused on optimizing RNA transfer into mature DCs using the method of electroporation and obtained high protein expression in 90% of mature DCs. Particular emphasis was placed on quantifying RNA transfer. Reconstitution of peptide-MHC (pMHC) ligands on RNA-pulsed DCs was measured with the help of effector-memory cytotoxic T lymphocytes (CTLs) specific for the melanoma-associated antigens tyrosinase and mutated cyclin-dependent kinase 4. In contrast to single-species RNA, transfer of native tumor-derived RNA or amplified tumor RNA into DCs resulted in very low or no capacity to reactivate these CTLs. A correlation was found between TAA message levels in tumor-derived RNA and pMHC ligand reconstitution on DCs. These results demonstrate that even TAAs with highly immunogenic mutated epitopes are not necessarily transferred when tumor-derived RNA is transfected into the DCs, thereby resulting in a lack of DC capacity to reactivate some preexisting effector-memory CTLs.

Cell Line, Tumor↗
PubMed1994

Altered queuine modification of transfer RNA involved in the differentiation of human K562 erythroleukemia cells in the presence of distinct differentiation inducers.

Altered queuine modification of tRNA has been correlated to neoplasia and cell differentiation, but much of the existing evidence is only circumstantial. In the present study, we used several distinct differentiation inducers to measure changes in Q-family tRNA species during the erythroid differentiation of human K562 erythroleukemia cells. Treatment of K562 cells with 3.6 microM 1-beta-D-arabinofuranosylcytosine (ara-C), 1 mM sodium butyrate, 0.1 mM hemin, or 5 microM 5-azacytidine resulted in growth inhibition, erythroid differentiation, and changes in queuine content of tRNA. In the presence of the irreversible inducer ara-C, the queuine content of tRNA increased markedly when the cells differentiated into benzidine-positive erythroid cells, and cell growth was inhibited. The increase in the queuine content of tRNA in differentiated K562 cells was an irreversible event. In cells incubated with the reversible inducer sodium butyrate, an increase in the queuine content of tRNA was correlated with the increase in benzidine-positive erythroid cells throughout the culturing period. After removal of the drug at 48 h, the queuine content of tRNA decreased concomitant with a decrease in benzidine-positive cells. Treatment with another reversible inducer, hemin, caused only a transient increase in the queuine content of tRNA, which was not correlated with the steady increase in benzidine-positive erythroid cells. The agent 5-azacytidine slightly inhibited cell growth but did not significantly change the percentage of benzidine-positive cells and the queuine content of tRNA. We further clarified the changes in queuine content of tRNA by analyzing the Q-containing isoacceptors of Q-family tRNA species including tRNA(Tyr), tRNA(His), tRNA(Asp), and tRNA(Asn) by RPC-5 chromatography, and found that the change in queuine content of tRNA(Tyr) was greater than the other Q-family tRNA species during induction by ara-C, sodium butyrate, and hemin. Our results indicate that the change in queuine content of tRNA is an irreversible event of terminal differentiation in ara-C induction and is a transient event of reversible differentiation in hemin induction. Sodium butyrate induction might represent a status between irreversible and reversible differentiation. Q-containing isoacceptors of tRNA might potentially play an important biological role during K562 cell differentiation.

Aspartate-tRNA Ligase↗
PubMed1995

Analysis of transfer RNA during the early embryogenesis of the freshwater teleost, Heteropneustes fossilis.

Total RNA as well as transfer RNA were quantified from mature ova apart from four different embryonic stages namely mid-cleavage, early gastrula, mid-gastrula and organogenesis of the freshwater teleost Heteropneustes fossilis. Total RNA as well as transfer RNA quantity follow a similar variation pattern, being maximum during mid-gastrulation. When analysed by total amino acid acceptance capacity, transfer RNA shows its maximum activity during mid-gastrulation. This coincides with the higher ratio of tRNA to total RNA at this stage. The relative aminoacylation capacity for Ser, Gly, Asn and Thr are found to be higher (9-34%) compared to that for other amino acids. Total tRNA, resolved into three peaks upon HPLC fractionation, shows a high cumulative peak area during mid-gastrulation and organogenesis. These results indicate a switch over of maternal to embryonic translation machinery during gastrulation.

Animals↗
PubMed1993

Fe.bleomycin cleaves a transfer RNA precursor and its "transfer DNA" analog at the same major site.

Previously, Fe.bleomycin (BLM) has been shown to mediate RNA cleavage in a fashion more highly selective than that of DNA. Because RNAs often assume secondary and tertiary structures not commonly encountered with DNAs, it was not clear whether the greater selectivity of RNA cleavage was a consequence of differences in the mononucleotide constituents of RNA and DNA, or of the three-dimensional structures of the individual substrates. Accordingly, we prepared a "tDNA" identical in sequence with Bacillus subtilis tRNA(His) precursor, the latter of which is known to be a good substrate for Fe(II).BLM A2 and which undergoes oxidative cleavage predominantly at U35. Remarkably, the tDNA underwent cleavage predominantly at T35. At higher concentrations of Fe(II).BLM A2, the tDNA was extensively degraded, while the tRNA(His) precursor was not. Competition experiments suggested that this was not due to more efficient binding of Fe.BLM to the tDNA; in fact the tRNA precursor appeared to be bound more efficiently. The lesser cleavage of the tRNA(His) may be due to limitations in the facility of chemical transformation following Fe.BLM binding, or else to the formation of RNA lesions that do not lead directly to RNA strand scission.

Bacillus subtilis↗
PubMed1994

Modulation of the suppression efficiency and amino acid identity of an artificial yeast amber isoleucine transfer RNA in Escherichia coli by a G-U pair in the anticodon stem.

The artificial amber suppressor corresponding to the major isoleucine tRNA from yeast (pVBt5), when expressed in E. coli, is a poor suppressor of the amber mutation lacIam181-Z. By analysing mutant forms, we could show that this was due to the presence of a U30-G40 wobble pair in the anticodon stem of the yeast tRNA and not to the level of the heterologously expressed tRNA. Efficient suppressors were obtained by restoring a normal U30-A40 or G30-C40 Watson-Crick pair. In vivo the mutant forms are exclusively charged by the bacterial lysyl-tRNA synthetase (LysRS), whereas the original yeast amber tRNA is charged at a low level by E. coli glutaminyl-tRNA synthetase (GlnRS) and LysRS. The inversion of the U30-G40 pair also induces a loss of the Gln identity. We conclude from these experiments that the U30-G40 base pair constitutes a negative determinant for LysRS interaction which operates either at the level of complex formation or at the catalytic step. As no direct contacts are seen between GlnRS and positions 30-40 of the complexed homologous tRNA, the U30-G40 pair of pVBt5 is believed to influence aminoacylation by GlnRS indirectly, probably at the level of the anticodon loop conformation by favouring an optimal apposition of the anticodon nucleotides with the protein.

Amino Acyl-tRNA Synthetases↗