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.     

Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids

Escherichia coli initiator methionine tRNA labeled in vivo with 5-fluorouracil (FUra) has been isolated and characterized. The tRNA, with essentially all its uracil and uracil-derived minor bases replaced by FUra, was purified by sequential chromatography, first on diethylaminoethylcellulose (DEAE-cellulose), at pH 8.9, followed by chromatography on Sepharose 4B, using a reverse salt gradient, then on DEAE-Sephadex A-50, and finally on benzoylated DEAE-cellulose. The last step resolved two FUra-substituted tRNAfMet-iso-accepting species, each with a specific activity over 1500 pmol/A260. Kinetic analysis shows both are aminoacylated at the same rate; apparent KmS for the two are 0.92 and 0.94 microM, compared with 1.7 microM for normal tRNAfMet. Chromatographic differences between the two forms of fluorinated tRNAfMet persist after aminoacylation, and the two tRNAs are not interconverted by denaturation and renaturation. The isoacceptors have nearly identical nucleoside composition, and both contain 7-methylguanosine and 2'-O-methylcytidine as the only modified nucleosides. Analysis of complete RNase T1 digests of the two methionine tRNAs shows that they differ in only one oligonucleotide. The sequence 20FpApGp, derived from the dihydrouridine loop and stem region, which is found in one of the isoaccepting forms of the tRNA, is replaced by an oligonucleotide containing adenine and guanine, but no FUra in the other. A modified FUra, with the properties of a 5-fluoro-5,6-dihydrouracil derivative, is detected in this tRNA. 19F NMR spectra of the two species of FUra-substituted initiator tRNA show 9-10 resolved resonances for the 12 FUra residues incorporated. The spectra differ primarily in the shift of one peak in the form lacking the sequence 20FpApGp, from 4.8 ppm downfield from free FUra (= 0 ppm) to 14.9 ppm upfield from the standard.     

19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

The 19F NMR spectrum of Escherichia coli tRNA1Val in which [5-19F]uridine replaces 93% of all uridine and uridine-derived residues has been examined at 93.6 and 235 MHz. The resolution of 11 peaks and visibility of two additional shoulders at either frequency for the 14 FUra residues in the molecule attests to the excellence of 19F as a probe for the structure of tRNA1Val in solution. No significant gain in resolution was attained at the higher frequency. A comparison of the relative areas in the different regions of the 19F spectrum of mixed [FUra]tRNAs with that of [FUra]tRNA1Val suggests that the three single resonances at lowest field in the region 86.5 to 88.5 ppm upfield from trifluoroacetate correspond to the three invariant bases which form tertiary hydrogen bonds in all tRNAs, namely, 8 (U or s4U), 54 (T), and 55 (phi) in unsubstituted tRNAs.     

Halogenated nucleic acids: effects of 5-fluorouracil on the conformation and properties of a polyribonucleotide and its constituents

The conformational properties of 5-fluorouracil derivatives are compared to uracil derivatives. FUrd, 5′-FUMP, and poly(FU) are studied as a function of pH and temperature by ¹⁹F- and ¹H-nmr spectroscopy, and the corresponding uracil derivatives by ¹H-nmr spectroscopy. FUrd exhibits no significant conformational changes with solution pH (5–10). In contrast, at low pH (6–7) 5′-FUMP and 5′-UMP show similar conformational features, while at high pH (9) 5′-FUMP shows significant conformational alterations. Also, poly(U) and poly(FU) are conformationally similar at low pH, but increasing pH induces changes in poly(FU). These changes are observed in the backbone [γ(C4′-C5′)], furanose, and furanose-base conformations. The apparent pK_a of N3-H ionization of the FUra base is determined by ¹H- and ¹⁹F-nmr to range from 7.5 to 8.2 [FUrd < 5′-FUMP < 5′-FUDP < poly(FU)]. These observations are interpreted as a result of electrostatic interactions generated between the ionized phosphate group and the negatively charged base moiety as the pH is raised. The interaction properties of poly(FU) with ApA are studied by ¹H- and ¹⁹F-nmr spectroscopy, and these properties compared to those published for poly(U). Poly(FU) forms a complex with ApA inducing upfield ¹H-shifts in both components, and downfield ¹⁹F- shifts in poly(FU). The base stoichiometry of the complex for poly(U)·ApA is 2U:1A at various U/A ratios. In contrast, the base stoichiometry of the poly(FU)·ApA complex appears to be dependent on the FU/A ratio. At high FU/A ratio, the complex is 2FU:1A, and as the FU/A ratio approaches unity the complex becomes 1FU:1A.     

Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted Escherichia coli transfer RNA

19F nuclear magnetic resonance has been used to study fully active Escherichia coli tRNA1Val in which 5-fluorouracil has replaced more than 90% of all uracil and uracil-derived modified bases. The 19F spectrum of the native tRNA contains resolved resonances for all 14 incorporated 5-fluorouracils. These are spread over a 6 ppm range, from 1.8 to 7.7 ppm downfield of the standard free 5-fluorouracil. The 19F resonances serve as sensitive monitors of tRNA conformation. Removal of magnesium or addition of NaCl produces major, reversible changes in the 19F spectrum. Most affected is the lowest field resonance (peak A) in the spectrum of the native tRNA. This shifts 2-3 ppm upfield as the Mg2+ concentration is lowered or the NaCl concentration is raised. Thermal denaturation of the tRNA results in a collapse of the spectrum to a single broad peak centered at 4.7 ppm. Study of the pH dependence of the 19F spectrum shows that five incorporated fluorouracils with 19F signals in the central, 4-5.5 ppm, region of the spectrum, peaks C, D, E, F, and H, are accessible to titration in the pH 4.5-9 range. All have pKa's close to that of free 5-fluorouridine (ca. 7.5). Evidence for a conformation change in the tRNA at mildly acidic pHs, ca. 5.5, is also presented. Four of the titratable 5-fluorouracil residues, those corresponding to peaks D, E/F, and H in the 19F spectrum of fluorine-labeled tRNAVal1, are essentially completely exposed to solvent as determined by the solvent isotope shift (SIS) on transfer of the tRNA from H2O to 2H2O. These are also the 5-fluorouracils that readily form adducts with bisulfite, a reagent that reacts preferentially with pyrimidines in single-stranded regions. On the basis of these results, resonances D, E, F, and H in the middle of the 19F spectrum are attributed to 5-fluorouracils in non-base-paired (loop) regions of the tRNA. Evidence from the ionic strength dependence of the 19F spectrum and arguments based on other recent studies with fluorinated tRNAs support earlier suggestions [Horowitz, J., Ofengand, J., Daniel, W. E., & Cohn, M. (1977) J. Biol. Chem. 252, 4418-4420] that the resonances at lowest field correspond to tertiary hydrogen-bonded 5-fluorouracils. Consideration of ring-current effects and the preferential perturbation of upfield 19F resonances by the cyclophotoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, which is known to react most readily with pyrimidines in double-stranded regions, permits initial assignment of upfield resonances to 5-fluorouracils in helical stems.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structure of aspartate transfer RNA from rabbit liver

The structure of rabbit liver aspartate tRNA₂ was derived by two postlabelling techniques involving labelling at 3′ or 5′ end followed by controlled hydrolysis with base-specific nucleases and product characterization by gel electrophoresis:

This tRNA of 76 residues contains 8 modified nucleotides (1-MeAdo, rThd, H₂Urd, Quo, two each of 5-MeCyd and ψrd). Although the proposed sequence resembles that of a recently described “major” isoacceptor of aspartate tRNA from rat liver, it differs in 13 nucleotides and contains an additional residue in the variable loop. Our tRNA sequence shows 65 per cent homology with an isoacceptor from the yeast, but only 55 per cent with the isoacceptor from Escherichia coli, and has very little resemblance to the aspartate isoacceptors from normal or tumor mitochondria.     

Chromosomal assignment of a large tRNA gene cluster (tRNALeu,tRNAGln, tRNALys,tRNAArg, tRNAGly) to 17p13.1

Summary A cluster of tRNA genes (tRNA _UAG ^Leu , tRNA _CUG ^Gln , tRNA _UUU ^Lys , tRNA _UCU ^Arg ) and an adjacent tRNA _GCC ^Gly have been assigned to human chromosome 17p12–p13.1 by in situ hybridization using a 4.2 kb human DNA fragment for tRNA^Leu, tRNA^Gln, tRNA^Lys, tRNA^Arg, and, for tRNA^Gly, 1.3 kb and 0.58 kb human DNA fragments containing these genes as probes. This localization was confirmed and refined to 17p13.100–p13.105 using a somatic cell hybrid mapping panel. Preliminary experiments with the biotiny lated tRNA Leu, Gln, Lys, Arg probe and metaphase spreads from other great apes suggest the presence of a hybridization site on the long arm of gorilla (Gorilla gorilla) chromosome 19 and the short arm of orangutan (Pongo pygmaeus) chromosome 19 providing further support for homology between HSA17, GGO19 and PPY19.     

The localization of tRNA 5 Asn,tRNAHis, and tRNAAla genes from Drosophila melanogaster by in situ hybridization to polytene salivary gland chromosomes

Transfer RNA _5; ^Asn , tRNA _; ^His , and tRNA^Ala were isolated from Drosophila melanogaster by means of Sepharose 4B chromatography and 2-dimensional polyacrylamide gel electrophoresis. The tRNAs were iodinated in vitro with Na¹²⁵I and hybridized in situ to salivary gland chromosomes from Drosophila. Subsequent autoradiography allowed the localization of the genes for tRNA _5; ^Asn in the regions 42A, 59F, 60C, and 84F; for tRNA^His in the regions 48F and 56E; and for tRNA^Ala in the regions 63A and 90C. From these and our previous results it can be concluded that the genes for the Q-base containing tRNAs (tRNA^Asn, tRNA^Asp, and tRNA^His, are not clustered in the Drosophila melanogaster genome.     

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

N⁶-Threonylcarbamoyl-adenosine (t⁶A) is a universal modification occurring at position 37 in nearly all tRNAs that decode A-starting codons, including the eukaryotic initiator tRNA (tRNA_i^Met). Yeast lacking central components of the t⁶A synthesis machinery, such as Tcs3p (Kae1p) or Tcs5p (Bud32p), show slow-growth phenotypes. In the present work, we show that loss of the Drosophila tcs3 homolog also leads to a severe reduction in size and demonstrate, for the first time in a non-microbe, that Tcs3 is required for t⁶A synthesis. In Drosophila and in mammals, tRNA_i^Met is a limiting factor for cell and animal growth. We report that the t⁶A-modified form of tRNA_i^Met is the actual limiting factor. We show that changing the proportion of t⁶A-modified tRNA_i^Met, by expression of an un-modifiable tRNA_i^Met or changing the levels of Tcs3, regulate target of rapamycin (TOR) kinase activity and influences cell and animal growth in vivo. These findings reveal an unprecedented relationship between the translation machinery and TOR, where translation efficiency, limited by the availability of t⁶A-modified tRNA, determines growth potential in eukaryotic cells.     

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA^Leu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA^Leu (CAG)—a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.     

Identification and codon reading properties of 5-cyanomethyl uridine,a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA₂^Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA₂^Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.     

Specific binding of bovine immunoglobulins to Sepharose and Sephadex

Bacteriophage T5 BglII/HindIII DNA fragment (803 basepairs), containing the genes for 2 tRNAs and 2 RNAs with unknown functions, was cloned in the plasmid pBR322. The analysis of DNA sequence indicates that tRNA genes code isoacceptor tRNAs^Ser (tRNA^Ser₁ and tRNA^Ser₂) with anticodons UGA and GGA, respectively. The main unusual structural feature of these tRNAs is the presence of extra non-basepaired nucleotides in the joinings of stem ‘b’ with stems ‘a’ and ‘c’.     

19F NMR studies of the binding of 5-fluoro-2′-deoxyuridylate to thymidylate synthase

Summary In the presence of thymidylate synthase, the ¹⁹F signal of 5-fluoro-2-deoxyuridylate is shifted upfield 0.6 ppm or 4.5 ppm depending on the enzyme preparation used. The bands at these positions represent different species of binary complex. When either binary complex is reacted with methylenetetrahydrofolate a ternary complex is formed with a ¹⁹F signal shifted 12.5 ppm upfield and broadened to 120 Hz.Substitution of the hydrogen atoms of the methylene group of methylenetetrahydrofolate with deuterium atoms results in line-narrowing of the spectrum of the ternary complex from 120 to 80 Hz indicating the close proximity of the methylene group to the fluorine atom in the ternary complex. A model compound, 5-fluoro-6-hydroxy-5-methyl-5, 6-dihydrouracil, gives a chemical shift in the same direction and of similar magnitude to that seen with the ternary complex.     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Two fractions of phenylalanine tRNA (tRNA^Phe₁ and tRNA^Phe₂) were purified by BD-cellulose and RPC-5 chromatography of crude tRNA isolated from barley embryos. Successive RPC-5 rechromatography runs of tRNA^Phe₂ showed its conversion into more stable tRNA^Phe₁, suggesting that the two fractions have essentially the same primary structure. Both tRNA^Phe₁ and tRNA^Phe₂ had about the same acceptor activity, but tRNA^Phe₂ was aminoacylated much faster than tRNA^Phe₁. RPC-5 chromatography of crude aminoacylated tRNA showed higher contents of phe-tRNA^Phe₂ than of phe-tRNA^Phe₁ but the ratio of these two fractions estimated by relative fluorescence intensity was about 1. Fluorescence spectra of tRNA^Phe from barley embryos suggest that it contains Y base similar to Y_w from wheat tRNA^Phe.