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PubMed1985

Characterization of the fluorescent bimane derivative of E. coli initiator transfer RNA (tRNAfMet).

The invariant modified base 4-thiouridine of the E. Coli initiator tRNA was chemically modified using a sulfhydryl specific fluorogenic probe, monobromobimane. The modified tRNAfMet is virtually indistinguishable biochemically from the native form in the aminoacylation and formylation reactions, and in its binding behavior to the ribosomal P site. Fluorescence quenching by I- increases 40% when the modified tRNA is charged with formylmethionine, even at this relatively well-shielded position in the tRNA elbow. Most important, the fluorescence polarization increases by a factor of 2, to almost the irrotational value, when fMet-tRNAfMet binds to the ribosomal P site, providing a useful tool for studying fMet-tRNAfMet-ribosome interaction equilibria and kinetics.

Bridged Bicyclo Compounds↗

PubMed1990

Effects of nucleotide- and aurodox-induced changes in elongation factor Tu conformation upon its interactions with aminoacyl transfer RNA. A fluorescence study.

The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNA(Phe)-Fl8, a functionally active analogue of Phe-tRNA(Phe) with a fluorescein dye convalently attached to the s4U-8 base. The association of EF-Tu.GDP with Phe-tRNA(Phe)-Fl8 resulted in an average increase of 33% in fluorescein emission intensity. This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu.GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu.GDP-containing ternary complex. The Kd for the Phe-tRNA(Phe)-Fl8.EF-Tu.GDP complex was found to average 28.5 microM, more than 33,000-fold greater than the Kd of the Phe-tRNA(Phe)-Fl8.EF-Tu.GTP complex under the same conditions. In terms of free energy, the delta G degree for ternary complex formation at 6 degrees C was -11.5 kcal/mol with GTP and -5.8 kcal/mol with GDP. Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu.GDP from the aa-tRNA on the ribosome. Aurodox (200 microM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 microM, and increased the Kd of the GTP complex by at least 6-fold. The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu.GDP affinity for aa-tRNA and also weakens EF-Tu.GTP affinity for aa-tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Bacterial Agents↗

PubMed1990

In vitro suppression of an amber mutation by a chemically aminoacylated transfer RNA prepared by runoff transcription.

An amber suppressor tRNA was prepared in vitro by runoff transcription with T7 RNA polymerase. Both full-length tRNA and truncated tRNA lacking the 3' terminal pCpA from the acceptor stem could be synthesized from the same DNA template. Truncated runoff suppressor tRNA could be enzymatically ligated to phenylalanyl-pCpA to generate aminoacylated full-length suppressor tRNA (Phe-tRNA(CUA)). Phe-tRNA(CUA) is capable of suppressing an amber (UAG) mutation in vitro with equivalent efficiency as suppressor prepared by anticodon-loop replacement of a naturally-isolated tRNA. The ease of suppressor tRNA preparation using this method, compared to anticodon-loop replacement, greatly facilitates the use of chemically acylated suppressor tRNA's for site-specifically incorporating unnatural amino acids into proteins.

Acylation↗

PubMed1991

Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp).

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.

Aspartate-tRNA Ligase↗

PubMed1994

Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites.

The number of tRNA binding sites in 80S ribosomes from Saccharomyces cerevisiae was assessed by means of tRNA saturation and translocation experiments. In the absence of cognate mRNA yeast ribosomes could bind 0.6 [32P]tRNA(Phe) per 80S while poly(U) programmed ribosomes accepted up to 1.7 tRNA(Phe) molecules per 80S or 0.5 molecules of Ac[14C]Phe-tRNA(Phe) per 80S. Compared with the known features of E. coli ribosomes these binding values indicated both the presence of three tRNA binding sites and the validity of the exclusion principle for peptidyl-tRNA binding to yeast ribosomes. Upon EF-2 dependent translocation of a complex containing deacyl-tRNA in the P-site and AcPHe-tRNA in the A-site, the deacylated tRNA does not leave the ribosome quantitatively. This observation suggests the presence of an E site in 80S ribosomes which is functionally equivalent to the one previously characterized in prokaryotic systems.

Binding Sites↗

PubMed1971

High-resolution x-ray diffraction patterns of crystalline transfer RNA that show helical regions.

Yeast phenylalanyl transfer RNA crystallizes in a simple orthorhombic unit cell (a = 33.2, b = 56.1, c = 161 A), and the crystal yields an x-ray diffraction pattern with a resolution of 2.3 A. From an analysis of the packing in the unit cell it is concluded that the molecular dimensions are approximately 80 by 33 by 28 A. The diffraction pattern viewed along the a-axis has a distribution characteristic of double-helical nucleic acids. However, this distribution is not found when the pattern is viewed along the b-axis. This has been interpreted as indicating that the double-helical portions of the transfer RNA molecule are approximately half a helical turn in length, and therefore can contain 4-7 base pairs. These results are consistent with the cloverleaf formulation of transfer RNA secondary structure.

Crystallization↗

PubMed1996

Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease.

OBJECTIVE: To identify and characterize a novel autoantibody, anti-WS, that binds total transfer RNA (tRNA). METHODS: Serum from patient WS, who had polyarthritis, Sjögren's syndrome, Raynaud's phenomenon, and interstitial pulmonary fibrosis, was used in this study. Characteristics of anti-WS and antibody-reactive determinants of tRNA were investigated by 32P immunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro. RESULTS: WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence. 32P immunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8S and 5S ribosomal RNAs from 32P-labeled HeLa cell extract. When deproteinized RNA was used as antigen source, total tRNAs were still precipitated by WS serum. An immunoprecipitation study, using various deletion mutants of Escherichia coli tRNA, demonstrated that both D and T psi C loops were needed for antibody binding. Substitution of nucleotide 18G with 18A of E coli tRNA(Trp), which is essential in the formation of the tertiary "L" shape of tRNA, inhibited binding by anti-WS antibodies. CONCLUSION: Anti-WS antibodies are novel autoantibodies directed against tRNAs. The antibody binding site is the common L-shaped tertiary structure conformed by the D loop and T psi C loop of tRNA, suggesting that the antibodies are induced by a conserved sequence among all species. Furthermore, these antibodies could be a marker for a newly recognized subset of connective tissue disease.

Autoantibodies↗

PubMed1969

N-formylseryl-transfer RNA.

The reactions between serine and transfer RNA from baker's yeast and from Escherichia coli have been investigated. Results obtained from in vitro, in vivo, and chemical syntheses and from electrophoretic, chromatographic, and radioautographic analyses demonstrate that N-formylseryl-transfer RNA is formed in these systems.

Autoradiography↗

PubMed1985

Crystallographic refinement of yeast aspartic acid transfer RNA.

The structure of yeast transfer RNA aspartic acid has been refined in one crystal form to 3 A resolution using the restrained least-squares method of Hendrickson and Konnert and real-space fitting using the FRODO program of Jones. The final crystallographic discrepancy index R is 23.5% for 4585 reflections with magnitudes twice their standard deviations between 10 and 3 A. With lower occupancies for some residues of the D-loop, the phosphate U1, and the base U33, the R-factor is 22.3%. The adaptation of the restrained least-squares program for nucleic acids and the progress of the refinement are described. The conformations are analysed with respect to stereochemistry and folding of the backbone. The contacts and hydrogen bonds of the secondary structure are compared with those of yeast tRNAPhe. The presence of only four bases in the variable loop, instead of five as in yeast tRNAPhe, leads to a rotation of residue 48 and a lateral movement of residue 46. These two rearrangements induce different environments for [U8 . . . A14] . . . A21 as well as for A9 and G45. Otherwise, all tertiary contacts observed in yeast tRNAPhe are present in yeast tRNAAsp, except for the absence of hydrogen-bonding between G18 of the D-loop and C56 of the T-loop. The presence of anticodon triplet pairing leads to a distribution of temperature factors different from that observed in yeast tRNAPhe with a stabilization of the AC stem-and-loop and a destabilization of the T and D-loops. We are inclined to suggest that the labilization of the interactions between the T and D-loops is a consequence of the interaction of the anticodon triplets of symmetry-related molecules through hydrogen bonding, which mimics the interaction between the anticodon and its cognate codon on the messenger RNA.

Anticodon↗

PubMed1977

Time dependence of ethionine-induced changes in rat liver transfer RNA methylation.

Methyl-deficient transfer RNA (tRNA) and subnormal levels of tRNA-methylating enzymes were found in the livers of female rats that had received injections of 250 mg DL-ethionine per kg body weight per day and 120 mg adenine per kg body weight per day for 2 days. Adenine alone had no effect. When the ethionine plus adenine injections were continued for longer periods of time, liver tRNA-methylating enzyme activity measured in vitro gradually increased and exceeded that of the controls. Concurrently, the relative methyl deficiency of liver tRNA decreased. The latter was evident because of the decreased ability of the tRNA to accept methyl groups during in vitro methylation catalyzed by homologous enzymes. Liver tRNA from animals that were treated with ethionine for 7 days could accept only about 40% as many methyl groups as could tRNA from animals that had received ethionine for only 2 days. No further significant change in methyl deficiency of the tRNA was seen when ethionine administration was extended to a total of 14 days. Enzyme preparations from ethionine-treated, but not control, rat livers contained dialyzable substances that inhibited the tRNA methylases and altered the base specificity of these enzymes. Although S-adenosylhomocysteine and S-adenosylethionine were found to be present in the liver preparations, neither of these substances could account for the observed changes in specificity.

Adenine↗

PubMed1989

Chromosomal assignment of a glutamic acid transfer RNA (tRNAGlu) gene to 1p36.

A gene for tRNAGlu has been assigned to human chromosome 1p36 by in situ hybridisation using a [3H]-labelled or biotinylated 2.4-kb (human) DNA fragment containing a tRNAGlu gene as a probe. With the biotinylated DNA probe a secondary statistically significant site of hybridisation was observed at 1q21-22 which might represent a pseudogene or related sequence. In fibroblasts from gorilla (Gorilla gorilla) using biotin labelling, a single site of hybridisation occurred at 1qter which provides further support for homology of 1q in the higher apes and human 1p.

Animals↗

PubMed1987

Mobility of individual 5-fluorouridine residues in 5-fluorouracil-substituted Escherichia coli valine transfer RNA. A 19F nuclear magnetic resonance relaxation study.

19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion. Measured relaxation parameters include the spin-lattice (T1) relaxation time at 282 MHz, the 19F(1H) NOE at 282 MHz, and the spin-spin (T2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz. Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T2. The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz. Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA(Val)1, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule. The dipolar relaxation data are analyzed by using the "two-state jump" and "diffusion in a cone" formalisms. Motional amplitudes (theta) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside. These amplitudes range from approximately 30 degrees to 60 degrees, assuming a correlation time (tau i,2) of 1.6 ns. By using available 19F n.m.r. assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA(Val)1, these motional amplitudes can be correlated directly with the environmental domain of the residue. Residues located in tertiary and helical structural domains show markedly less motion (theta approximately equal to 30 to 35 degrees) than residues located in loops (theta approximately equal to 45 to 60 degrees). A correlation between residue mobility and solvent exposure is also demonstrated. The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA(Phe).

Chemical Phenomena↗

PubMed1990

Enzymatic addition of guanylate to histidine transfer RNA.

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Animals↗

PubMed1982

Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver.

The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteine, the most highly purified 75Se-fractions were greater than 100-fold purified relative to 35S. These fractions contained less than 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.

Animals↗

PubMed1988

Interactions of porphyrins and transfer RNA.

The interactions of the free base porphyrin, tetra-(4N-methylpyridyl)porphyrin and its copper(II), manganese(III) and zinc(II) complexes with brewer's yeast type V phenylalaninyl tRNA were evaluated by UV-visible spectroscopy, circular dichroism and melting temperature studies over a range of magnesium ion concentrations and ionic strengths. Scatchard analysis of absorption spectra of the porphyrins in the presence of tRNA showed the free base, copper and zinc porphyrins to have binding constants of 7.3 X 10(7), 1.7 X 10(6) and 2.3 X 10(8), respectively; the manganese(III) complex did not demonstrate changes in its electronic spectra that enable the calculation of a binding constant. The results of the spectroscopic studies indicate a mode of binding for the free base, copper(II) and zinc(II) complexes that is neither intercalative nor simply outside electrostatic. The magnitude of the binding constants and the UV-visible results support intercalation, but the analyses of the thermal denaturation studies and the circular dichroism evaluations suggest that the porphyrins are associating at a single site in a fold of the tertiary structure of the tRNA close to several crucial hydrogen bonds, perhaps in the vicinity of the P10 loop. That the manganese(III) complex does not bind in this site points to constraints on the axial thickness of a molecule that may be accommodated in this locus.

Cations, Divalent↗

PubMed1989

Studies of interactions of porphyrins with transfer RNA by high-resolution NMR.

The interactions of tetra-4N-methylpyridyl porphyrin and its zinc(II), copper(II) and manganese(III) complexes with brewer's yeast type V phenylalanine specific tRNA have been evaluated by high-resolution NMR. Differences in chemical shifts have been noted for three proton resonances in response to the presence of small quantities of the free base and the zinc and copper complexes. The protons giving rise to these signals are located on bases T54 and psi 55, both of which are involved in the primary intraloop and interloop hydrogen bonds that hold the D and T psi C loops together in the tertiary structure. In addition, broadening of specific resonances due to hydrogen bonding protons in the D stem at low ratios of porphyrin to tRNA indicates that the association of porphyrins increases the rate of imino proton exchange. The titration of the tRNA with the manganese(III) complex did not reveal shifts or specific broadening comparable to the other porphyrins at low ratios. The changes induced in the NMR spectrum of tRNA by porphyrins define their site of interaction with the polynucleotide. This site, at the outside of the elbow-bend in the tRNA 'L', is different from the locus of binding in tRNA for other classical DNA intercalators. Furthermore, a new mode of binding may be involved that is neither intercalative nor simply electrostatic.

Binding Sites↗

PubMed1988

Transcription of the fimbrial subunit gene and an associated transfer RNA gene of Pseudomonas aeruginosa.

Gene fimA encoding the structural subunit of the fimbriae of Pseudomonas aeruginosa PAK is located in the centre of a 1.2-kb HindIII genomic DNA fragment [see also Sastry et al., J. Bacteriol. 164 (1985) 571-577], which in turn is located within a 6.2-kb EcoRI fragment. Immediately downstream from fimA is a putative threonine tRNA gene [Dalrymple and Mattick, Biochem. Int. 13 (1986) 547-553]. Northern blotting experiments showed that fimA is transcribed to an mRNA of approx. 650 nucleotides, which also includes the threonine tRNA sequence but no other protein-coding region. There was no indication that this mRNA is processed to release the tRNA sequence. However, the tRNA did appear to be expressed independently from its own promoter in the region 3' to fimA. When these sequences were introduced into Pseudomonas putida, we found that the level of expression of fimA from the cloned 6.2-kb EcoRI fragment was approx. 30-fold greater than that from the smaller HindIII fragment, whereas that of the specific tRNA species was unaltered. The size of the fimA transcript was also unaltered. These results provide evidence that the fimA gene is subject to specific transcriptional activation in vivo and that this activation involves sequences flanking the 1.2-kb HindIII fragment.

Bacterial Proteins↗

PubMed1987

Internal motions in yeast phenylalanine transfer RNA from 13C NMR relaxation rates of modified base methyl groups: a model-free approach.

Internal motions at specific locations through yeast phenylalanine tRNA were measured by using nucleic acid biosynthetically enriched in 13C at modified base methyl groups. Carbon NMR spectra of isotopically enriched tRNA(Phe) reveal 12 individual peaks for 13 of the 14 methyl groups known to be present. The two methyls of N2,N2-dimethylguanosine (m22G-26) have indistinguishable resonances, whereas the fourteenth methyl bound to ring carbon-11 of the hypermodified nucleoside 3' adjacent to the anticodon, wyosine (Y-37), does not come from the [methyl-13C]methionine substrate. Assignments to individual nucleosides within the tRNA were made on the basis of chemical shifts of the mononucleosides [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. A., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408; Smith, C., Schmidt, P. G., Petsch, J., & Agris, P. F. (1985) Biochemistry 24, 1434-1440] and correlation of 13C resonances with proton NMR chemical shifts via two-dimensional heteronuclear proton-carbon correlation spectroscopy [Agris, P. F., Sierzputowska-Gracz, H., & Smith, C. (1986) Biochemistry 25, 5126-5131]. Values of 13C longitudinal relaxation (T1) and the nuclear Overhauser enhancements (NOE) were determined at 22.5, 75.5, and 118 MHz for tRNA(Phe) in a physiological buffer solution with 10 mM MgCl2, at 22 degrees C. These data were used to extract two physical parameters that define the system with regard to fast internal motion: the generalized order parameters (S2) and effective correlation times (tau e) for internal motion of the C-H internuclear vectors.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticodon↗