Nucleosides. 145. Synthesis of 2,5′-Anhydro-2-Thiouridine and its Conversion to 3′-O-Acetyl-2,2′-Anhydro-5′-Chloro-5′-Deoxy-2-Thiouridine. Studies Directed Toward the Synthesis of 2′-Deoxy-2′-Substituted arabino Nucleosides (7)

Abstract

In an attempt to introduce a substituent at C-2′ in the “up” arabino configuration directly by nucleophilic displacement reaction of a preformed pyrimidine ribonucleoside, we synthesized 2,5′-anhydro-5′-deoxy-2-thiouridine (6) in three steps from uridine. Compound 6 was converted into the 3′-O-acetyl derivative 7. Upon treatment of 7 with triflyl chloride in methylene chloride in the presence of triethylamine and p-dimethylaminopyridine, 2,2′-anhydro-1-(3-O-acetyl-5-chloro-2,5-dideoxy-β-D-arabinofuranosyl)-2-thiouracil (9) was obtained as the only isolable product. Obviously, the intermediate 3′-O-acetyl-2,5′-anhydro-2′-O-triflyl-2-thiouridine (8) was attacked by the chloride nucleophile at C-5′ first giving the 2′-O-triflyl-2-thiouridine intermediate from which 9 was formed by intramolecular nucleopilic reaction.     

Photolabile and paramagnetic derivatives of the nucleoside X and of Escherichia coli tRNAPhe.

The synthesis of N3-[3-L-(5-azido-2-nitrobenzamido)-3-carboxypropyl]uridine (4b) and N3-[3-carboxy-3-L-(2,2,5,5-tetramethyl-3-pyrroline-3-carbonylamino)propyl]uridine Npyr-oxyl (4c) starting from the nucleoside X (4a) and the appropriate N-hydroxysuccinimide ester 1 or 2 is described. After acylation of tRNAPhe from E. coli (5a) with 1 or 2, the photolabile tRNAPhe derivative 5b and the paramagnetic tRNAPhe derivative 5c could be isolated. The position of modification in the polynucleotide chain was elucidated by comparison of the ribonuclease II/alkaline phosphatase digestion products of the substituted and unsubstituted tRNAPhe samples, and was identified as being exclusively the amino group of the nucleoside X in position 47 of E. coli tRNAPhe.     

Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli.

N4-Aminocytidine, a nucleoside analog, is strongly mutagenic to various organisms including Escherichia coli. Using E. coli WP2 (trp), we measured the incorporation of [5-3H]N4-aminocytidine into DNA and at the same time measured the frequency of reversion of the wild type, thereby attempting to correlate the incorporation with mutation induction. First, we observed that N4-aminocytidine uptake by the E. coli cells was as efficient as cytidine uptake. High-pressure liquid chromatographic analysis of nucleoside mixtures obtained by enzymatic digestion of isolated cellular DNA showed that the DNA contained [3H]N4-aminodeoxycytidine, corresponding to 0.01 to 0.07% of the total nucleoside; the content was dependent on the dose of N4-aminocytidine. There was a linear relationship between the N4-aminocytosine content in DNA and the mutation frequency observed. These results constitute strong evidence for the view that the N4-aminocytidine-induced mutation in E. coli is caused by the incorporation of this agent into DNA as N4-aminodeoxycytidine. We also found that the major portion of radioactivity in DNA of cells that had been treated with [5-3H]N4-aminocytidine was in the deoxycytidine fraction. We propose a metabolic pathway for N4-aminocytidine in cells of E. coli. This pathway involves the formation of both N4-aminodeoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate; the deoxycytidine 5'-triphosphate formation is initiated by conversion of N4-aminocytidine into uridine. In support of this proposed scheme, a cytidine deaminase preparation obtained from E. coli catalyzed the decomposition of N4-aminocytidine into uridine and hydrazine.     

Reaction of tRNAPhe from yeast with 1-fluoro-2,4-dinitrobenzene. Attachment sites of the potential antigenic-determining 2,4-dinitrophenyl residues.

The reaction of 1-fluoro-2,4-dinitrobenzene with tRNAPhe from yeast, for the introduction of antigenic-determining 2,4-dinitrophenyl residues into tRNA, took place only at adenosine residues in tRNAPhe. After reaction at pH 8.0 and 50 degrees C two kinds of products were detected: one was ribose-modified adenosine which was derived from the 3' terminus of tRNA, and the other was base-modified adenosine. The sites and extent of the modification of each particular adenosine residue of tRNAPhe were determined as follows: 5 (6% modified), 31 (2%), 35 (36%), 67 (5%), and 76 (51%). Thus mainly the terminal adenosine and one adenosine in the anticodon loop bear the 2,4-dinitrophenyl residue.     

Ability of modified forms of phenylalanine tRNA to stimulate guanosine pentaphosphate synthesis by the stringent factor-ribosome complex of E. coli

tRNA(Phe) of E. coli, modified at its 4-thiouridine ((4)Srd) and 3-(3-amino-3-carboxypropyl)uridine (nbt(3)Urd) residues, was tested for its ability to induce (p)ppGpp synthesis. The (4)Srd residue was derivatized with the p-azido-phenacyl group, cross-linked to Cyd(13), and the borohydride reduction product of the cross-link was prepared. The nbt(3)Urd residue was derivatized with the N-(4-azido-2-nitrophenyl)glycyl group. None of these derivatives had more than a minor effect on the affinity of the tRNA for the stringent factor-ribosome complex, and no effect at all on the maximum velocity of (p)ppGpp synthesis, either at 2 or 82 mM NH(4)Cl. These two regions of the tRNA which are on opposite faces of the tRNA molecule do not appear to be structurally important for recognition by the stringent factor-ribosome complex. They may provide useful sites, therefore, for the introduction of photoaffinity or fluorescent probes with which to study tRNA-stringent factor recognition.     

Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA. Its use for E. coli ribosome structural analysis

In vivo incorporation of the uridine-photoactivable analogue, 4-thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated. It is highly dependent on the exogenous uridine and 4-thiouridine concentrations as well as on temperature. We have defined conditions allowing the substitution of 13 +/- 2% of the uridine residues in bulk RNA by 4-thiouridine. On a high-Mg2+ sucrose gradient, 33 +/- 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non-associated 50S and 30S particles containing immature rRNA. The thiolated 70S ribosomes tolerate a 4-5% substitution level (40 thiouridine molecules/particle). Surprisingly, 3-4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4-thiouridine-substituted rRNA. Specific 366-nm photoactivation increased this proportion to 10-12%, i.e. up to six or seven ribosomal protein molecules/particle. The photochemical cross-linking proceeds with apparent first-order kinetics with a quantum yield close to 5 X 10(-3). Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross-linking were independent of oxygenation conditions. The thiolated (4.5%) 70S ribosomes allowed the poly(U)-directed poly(Phe)synthesis at 48% the control rate. Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively.     

Carbon-13 NMR studies on [4-13C] uracil labelled E. coli transfer RNA1(Val1).

In this paper we describe carbon-13 nuclear magnetic resonance results on 13C-enriched purified transfer RNAI(VAL) from from E. coli SO-187, a uracil requiring auxotroph. The organism was grown on uracil 90% 13C-enriched at the carbonyl C4 position. Transfer RNAI(Val) was purified from bulk tRNA by sequential chromatography on columns of BD cellulose, DEAE-Sephadex A-50 and reverse gradient sepharose 4B. Dihydrouridine, 4-thiouridine, and uridine 5-oxyacetic acid located at discrete positions in the polymer backbone were tentatively assigned in the highly resolved 25 MHz 13C-spectra. Chemical shift versus temperature plots reveal differential thermal perturbation of the ordered solution structure, evident in the large dispersion (ca 3-4 ppm) of the uridine C4 resonances. Over the range 26-68 degrees C, V in the anticodon displays the largest downfield shift. Whereas several uridine residues rapidly shift downfield between 50-68 degrees, one moves upfield beginning at 37 degrees. The results are qualitatively compared with proton NMR analysis of the three dimensional structure.     

15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.     

Isolation and characterization of 5-carbamoylmethyluridine and 5-carbamoylmethyl-2-thiouridine from human urine

Two new modified uracil nucleosides, 5-carbamoylmethyuridine (ncm5U, I) and 5-carbamoylmethyl-2-thiouridine (ncm5s2U, II) were isolated from a 24 hr collection of a normal human urine. The structures were assigned on the basis of UV, NMR and mass spectral data and confirmed by comparison of the spectral data and HPLC mobilities with those of authentic samples. On the basis of experimental data it appears possible that 5-carbamoylmethyl-2-thio-uridine (ncm5s2U, II) may be a degradation product produced from a labile precursor by the chemical treatments during the isolation procedure. However, the other nucleoside (ncm5U,I) certainly appears to be of metabolic origin and was also found in the urines of one chronic myelogenous leukemia and one lung carcinoma patient. Abbreviations used are: tRNA-transfer ribonucleic acid, TMS-trimethylsilyl, RP-HPLC--reverse phase high performance liquid chromatography, EI--electron impact, cm5U-5-carboxymethyluridine, mcm5U-5-methoxycarbonylmethyluridine, cm5s2U-5-carboxymethyl-2-thiouridine, mcm5s2U-5-methoxycarbonylmethyl-2-thiouridine, t6A-9-beta-D-ribofuranosyl-[N(purin-6-yl)carbamoyl]-1-threonine, C-cytidine, acp3u-3-(3-amino-3-carboxypropyl)uridine, AICR-aminoimidazole carboxamide riboside, alpha-4-PCNR & beta-4-PCNR-9-alpha-D-(or beta-D)-ribofuranosyl-pyridin-4-one-3-carboxamide, H x 7R-7-beta-D-ribofuranosyl hypoxanthine, m3U-3-methyluridine, m1I-1-methylinosine, m1G-1-methylguanosine, DI-5'-deoxyinosine, dms5OA-5'-deoxy-5'-methylthioadenosine sulfoxide, m2(2)G-N2-dimethylguanosine, psi-psi-uridine, A-adenosine, I-inosine, CML-chronic myelogenous leukemia mam5s2U-5-methylaminomethyl-2-thiouridine, ncm5U-5-carbamoylmethyluridine, ncm5s2U-5-carbamoylmethyl-2-thiouridine, UV-ultraviolet, NMR-nuclear magnetic resonance, HPLC-high performance liquid chromatography, GC-MS-gas chromatography-mass spectrometry.     

Expression in Escherichia coli, refolding, and purification of the recombinant mature form of human matrix metalloproteinase 7 (MMP-7)

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2).     

Photochemical reactions of 4-thiouridine disulfide and 4-benzylthiouridine--the involvement of the 4-pyrimidinylthiyl radical.

The reactions of a disulfide and a benzylsulfide derived from 4-thiouridine were studied in aqueous acetonitrile using stationary and laser flash photolysis methods. Irradiation of the compounds results in specific cleavage of the S-S bond in the disulfide and the S-CH(2) bond in the sulfide. Identical pyrimidine-derived intermediates were observed in the transient absorption spectra (lambda(max) = 420 nm, epsilon(max) approximately 2500 M(-1) cm(-1)) recorded for both compounds in laser flash photolysis experiments. The intermediate was identified as the 4-pyrimidinylthiyl radical. Irradiation of the disulfide in the absence of oxygen gives 4-thiouridine while the sulfide under identical conditions produced, additionally, 3-benzyl-4-thiouridine as a stable photoproduct. The formation of the latter photoproduct provides evidence for the existence of the N-centered 4-thioxopyrimidynyl radical formed from the initially produced S-centered (thiyl) radical. The 4-thiouridine is formed from the radicals generated in the primary photochemical step by an H abstraction reaction from the solvent (acetonitrile) or from additives (alcohols) that were purposely added. Interestingly, in contrast to the benzylsulfide, the photoreaction of the disulfide is quenched by molecular oxygen with the concomitant formation of uridine. However it appears that uridine is not produced as a result of the reaction of the radicals with oxygen. A mechanism is proposed for the photochemical transformations of the disulfide and benzylsulfide derived from 4-thiouridine. The proposed mechanism is based on the structures of the identified stable photoproducts, the values of the photoreaction quantum yields determined under differing irradiation conditions, and the flash photolysis results.     

Analogs of methionyl-tRNA synthetase substrates containing photolabile groups.

Three photolabile analogs of substrates of methionyl-tRNA synthetase were synthesized. In one, the 4-thiouridine at the 8 position of E. coli tRNAfMet was alkylated with [14C]p-azidobromoacetanilide. In the second, [14C]p-azidobenzoic acid hydrazide was condensed with the 3'-terminal dialdehyde of periodate-oxidized Escherichia coli tRNAfMet. The modified tRNAs could be purified by chromatography on benzoylated DEAE-cellulose. The third photolabile compound was [3H]methioninyl-8-azido-adenosine 5'-phosphate, an analog of the methionyl adenylate intermediate in the aminoacylation reaction. Irradiation of each of these compounds in the presence of equimolar amounts of E. coli methionyl-tRNA synthetase of micrometer concentrations gave 5-15% crosslinking.     

Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies

Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2′-Deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2′-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2′-deoxyinosine 9 was prepared from 2′-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-¹⁵N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-β-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.     

Novel E. coli mutants deficient in biosynthesis of 5-methylaminomethyl-2-thiouridine.

Novel E. coli mutants deficient in biosynthesis of 5- methylaminomethyl -2-thiouridine were isolated based on a phenotype of reduced readthrough at UAG codons. They define 2 new loci trmE and trmF , near 83' on the E. coli map. These mutants are different from strains carrying trmC mutations, which are known to confer a methylation deficiency in biosynthesis of 5- methylaminomethyl -2-thiouridine. tRNA from mutants carrying trmE or trmF mutations was shown to carry 2-thiouridine instead of 5- methylaminomethyl -2-thiouridine. This deficiency affects the triplet binding properties of the mutant tRNA. Our results suggest that the 5- methylaminomethyl group stabilizes the basepairing of this modified nucleotide with G, most likely through direct interaction with the ribosomal binding site(s).     

Synthetic study on carbocyclic analogs of cyclic ADP-ribose, a novel second messenger: an efficient synthesis of cyclic IDP-carbocyclic-ribose.

An efficient synthesis of cyclic IDP-carbocyclic-ribose, as a stable mimic for cyclic ADP-ribose, was achieved. 8-Bromo-N1-carbocyclic-ribosylinosine derivative 10, prepared from N1-(2,4-dinitrophenyl)inosine derivative 5 and an optically active carbocyclic amine 6, was converted to 8-bromo-N1-carbocyclic-ribosylinosine bisphosphate derivative 15. Treatment of 15 with I2 in the presence of molecular sieves in pyridine gave the desired cyclic product 16 quantitatively, which was deprotected and reductively debrominated to give the target cyclic IDP-carbocyclic-ribose (3).     

禽流感病毒H5N1血凝素基因和神经氨酸酶基因在大肠杆菌中的表达

目的：克隆、表达和鉴定禽流感病毒H5N1血凝素基因（hemagglutinin,HA）和神经氨酸酶基因（neuramidinase,NA）序列,为制备抗体和基因工程疫苗打下基础。方法：在成功克隆禽流感病毒H5N1全长HA、NA基因并测序的基础上,将部分基因序列克隆到表达载体pET32a（＋）上,全基因序列克隆到表达载体pGEX4T-1上,构建了重组表达质粒pET32a（＋）/HA(49~1587bp)、pET32a（＋）/NA(121~1141bp)、pGEX4T-1/HA、pGEX4T-1/NA,转化大肠杆菌BL21/rosetta,IPTG诱导表达,利用Ni2＋亲和层析柱和GSTra P4B亲和层析柱对重组蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。结果：重组蛋白在大肠杆菌中可以高效达,SDS PAGE显示其相对分子质量与预计大小一致,蛋白纯度占总蛋白的90％以上。ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。结论:成功克隆和表达了禽流感病毒H5N1 HA、NA基因序列,为禽流感病毒H5N1诊断试剂和疫苗的开发等进一步的研究奠定了基础。     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings VI. Anabolic Pathway of 4-Thiouridine

In germinating radish seeds, [U-¹⁴C]-4-thiouridine was convertedto 4-thio-UMP, 4-thio-UDP, 4-thio-UTP, 4-thio-UDP glucose and4-thiouracil, of which 4-thiouracil accounted for 60–85%.4-Thio-UTP is incorporated into RNAs of radish seedlings [Shibataet al. (1980) FEBS Lett. 119: 85]. These same metabolites werelabeled following germination of radish seeds with [2-¹⁴C]-4-thiouracil.4-Thiouridine was hydrolyzed by the uridine nucleosidase (EC3.2.2.3 [EC] ) of radish seedlings as effectively as was uridine.The activity of uridine nucleosidase was increased by germinationwith 4-thiouridine. These results are a strong indication that4-thiouridine is converted to 4-thiouracil, then to 4-thio-UMPby uracil phosphoribosyltransferase (EC 2.4.2.9 [EC] ). The alternativeformation of 4-thio-UMP from 4-thiouridine by uridine kinase(EC 2.7.1.48 [EC] ) also was suggested. A possible mechanism whichmay cause inhibition of chloroplast biogenesis in 4-thiouridine-culturedseedlings is discussed. (Received October 12, 1981; Accepted January 14, 1982)     

The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function.

The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.     

Coprecipitation of active uridine kinase and phosphomonoesterase from rat kidney by Zn2+-ions. III. Enzymes relevant to cancer chemotherapy.

Partially purified enzyme fraction from rat kidney possessing high uridine kinase and phosphomonoesterase activity was insolubilized by means of zinc precipitation without substantial loss of the activity. While uridine kinase in a soluble and Zn-precipitated form was inhibited by low concentrations (0.5-1.0 mM) of Zn2+-ions, phosphomonoesterase was fully active. In contrast to the soluble fraction, the two enzymes in zinc-precipitated and lyophilized preparations were stable on heating at 100 degrees C. Metal complexed proteins catalyze the dephosphorylation of 5'-UMP, 6-AzaUMP as well as of 2'(3')-UMP or 2,4-dinitrophenyl phosphate indicating thus the presence of several phosphomonoesterases in the complex.     

Lanthanide fluorescence studies of transfer RNAf(met) conformation.

The possible difference in conformation between aminoacylated and deacylated tRNA is examined using the optical and photochemical properties of the 4-thiouridine residue of E coli tRNAf(Met). No differences were seen between fMet-tRNAf(Met) and tRNAf(Met) observing the native fluorescence of 4-thiouridine, energy transfer from 4-thiouridine to the bound lanthanide ions, Tb3+ or Eu3+, or the rates of the photochemical cross-linking reaction of 4-thiourdine. While these results do not necessarily mean that there is no conformational difference between the aminoacylated and deacylated species, they do restrict the possible nature and magnitude of any conformational difference between the two species. In addition, preliminary thermal denaturation studies of tRNAf(Met), monitoring 4-thiouridine emission and energy transfer to Tb3+, indicate an unexplained melting phenomenon near 25 degrees C in the presence of Mg2+.