Recombinant photoreactive tRNA molecules as probes for cross-linking studies

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.     

Improved separation of modified nucleosides from tRNA hydrolysates: the patterns of tRNA methylation in rat tissues

A sensitive and reproducible method for the isolation of minor nucleosides derived from tRNA is described. The nucleosides obtained from enzymatic digestion of tRNA are separated into several groups using a QAE Sephadex column and increasing concentrations of boric acid in a step-wise manner. The nucleosides in each group are separated by isocratic elution from a preparative Partisil 10-SCX column and high-performance liquid chromatography at ambient temperature. With this method we have determined the patterns of tRNA methylation in vitro with extracts from rat bone, liver, kidney and adrenal glands. Although different tissues appear to contain the same tRNA methyltransferases, the patterns of methylated nucleosides are different.     

Metabolism of tRNA in rats with aflatoxin B1-induced hepatomas

This study describes effects of aflatoxin B1-induced hepatomas on RNA metabolism in rats. At 4 and 24 hours after the administration of L-(14CH3)-methionine, tRNA was isolated from the livers and hydrolyzed enzymatically to nucleosides which were quantitatively measured by HPLC. Radioactivity of the nucleosides was also determined. The data indicate that although tRNA methylation may be more rapid in livers with hepatomas, catabolism of tRNA in tumorous tissue is slower than in control livers. The large increase in some radioactive methylated nucleosides and bases by the tumor-bearing rats during the 24-hour period following the administration of labeled methionine indicates increased turnover of mRNA and rRNA as well as tRNA. Since degradation of tumor tRNA appears to be delayed, the excessive amounts of the urinary methylated nucleosides must be derived from RNA in nonneoplastic tissue.     

Synthesis of RNA containing O-beta-D-ribofuranosyl-(1'-2')-adenosine-5'-phosphate and 1-methyladenosine, minor components of tRNA

tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is well-known based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-beta-D-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5'-O-phosphorylated 2'-O-(beta-D-ribofuranosyl)-beta-D-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA(Met) will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.     

Determination of DNA base composition by reversed-phase high-performance liquid chromatography

Abstract DNA base composition was determined by reversed-phase high-performance liquid chromatography (HPLC). DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase. The mixture of nucleosides was applied to HPLC without any further purification. One determination by chromatography needed 2 μg of hydrolysed nucleosides and took only 8 min. The relative standard error of nucleoside analysis was less than 1%. The system described here gives a direct and precise method for determining DNA base composition.     

Quantitative determination of transfer RNA base composition using high-pressure liquid chromatography

An analytical method is presented for the quantitative determination of certain major and modified bases in unfractionated rat liver transfer RNA (tRNA), tRNA was hydrolyzed with perchloric acid, and the liberated bases were separated by high-pressure liquid chromatography. Bases were selectively detected in tRNA hydrolysates at wavelengths near their uv-absorption maxima. Recovery values for individual bases generally were in the 80–100% range. The composition of rat liver tRNA with respect to 10 bases was determined, and the levels of these bases were in agreement with published values determined by other methods.     

Identification of modified nucleosides in intact transfer ribonucleic acid by pyrolysis-electron impact-collisional activation mass spectrometry.

A novel mass spectrometric method has been developed for the detection and identification of dihydrouridine, ribothymidine, 4-thiouridine, and 7-methylguanosine in Escherichia coli tRNAs. The method utilizes (a) Pyrolysis-Electron Impact-Mass Spectrometry (PYEIMS), a procedure which releases the purine and pyrimidine bases from the intact, underivatized tRNA molecule. The mass spectrum exhibits intense peaks for the bases deriving from the common nucleosides in tRNA as well as peaks of much lower intensity at mass values expected for the bases from modified components known to be present in the tRNA; and, (b) Collisional Activation Mass Spectrometry (CAMS), a technique which permits the isolation of a single ion species from a complex mass spectrum. Subsequent fragmentation of that species yields a characteristic collisional activation spectrum. Such analyses of the ion species that were presumed to originate from H2Urd, rThd, 4SUrd, and 7MeGuo in the tRNA were used to define the structure and, thus, the identity of each component. Attributes of the PYEICAMS technique are that (a) precise structural elucidation of minor nucleosides present in tRNAs at the 1 - 4% level is obtained; (b) the high order of sensitivity allows the analysis to be done on microgram amounts of tRNA; and (c) there is no requirement for enzymatic or chemical hydrolysis of the tRNA or for subsequent chromatographic separation methods.     

Maturation of a hypermodified nucleoside in transfer RNA.

E. coli C6 rel- met- cys- was cultured in a fully supplemented medium and in media lacking cysteine or methionine. tRNA isolated from the three cultures containted, respectively, a normal complement of modified nucleosides; a deficiency in thiolated nucleosides and a deficiency in methylated nucleosides. Both sulfur-deficient tRNA and methyl-deficient tRNA contained large amounts of N-6- (delta-2-isopentenyl) adenosine and small amounts of the 2-methylthio derivative. Methyl-deficient tRNA contained, in addition a large amount of a cytokinin active, differently modified nucleoside that is believed to be a sulfur derivative of N6-(delta-2-isopentenyl) adenosine. The structure of this compound is unknown. When methly-deficient tRNA and the precusor the tRNA-Tyr su3-+ A25 were enzymatically methylated in vitro, methyl groups were incorporated into derivatives of isopentenyladenosine. These results indicate that the biosynthesis of the 2-methylthio derivative of isopentenyladenosine may occur in a sequential manner, i.e., thiolation of isopentenyladenosine followed by methylation.     

A "gain of function" mutation in a protein mediates production of novel modified nucleosides

The mutation sufY204 mediates suppression of a +1 frameshift mutation in the histidine operon of Salmonella enterica serovar Typhimurium and synthesis of two novel modified nucleosides in tRNA. The sufY204 mutation, which results in an amino-acid substitution in a protein, is, surprisingly, dominant over its wild-type allele and thus it is a "gain of function" mutation. One of the new nucleosides is 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modified by addition of a C(10)H(17) side chain of unknown structure. Increased amounts of both nucleosides in tRNA are correlated to gene dosage of the sufY204 allele, to an increased efficiency of frameshift suppression, and to a decreased amount of the wobble nucleoside mnm(5)s(2)U34 in tRNA. Purified tRNA(Gln)(cmnm(5)s(2)UUG) in the mutant strain contains a modified nucleoside similar to the novel nucleosides and the level of aminoacylation of tRNA(Gln)(cmnm(5)s(2)UUG) was reduced to 26% compared to that found in the wild type (86%). The results are discussed in relation to the mechanism of reading frame maintenance and the evolution of modified nucleosides in tRNA.     

Photo-emissions of nucleic acids and related compounds in the 120--250 nm region]

Photoemission of thin films of uracil, citosine, timine, adenine, quanine, DNA, RNA, nucleosides, nucleotides in the spectral region 125-250 nm is investigated. Photoemission work functions are determined, their values are in the interval of 5.7-6.1 ev. The dependence of photoemission efficiency on wave length for uracil, timine, DNA, RNA are measured. The results of this work with earlier data on luminescence yields are discussed. Possibility of radiation recombination process for wave lengths shorter than 160 nm is suggested.     

A rapid assay technique for RNA ribose methylases.

A rapid technique for quantitative separation of ribose-methylated nucleosides from base-methylated and non-methylated nucleosides by chromatography on DEAE-cellulose paper in the presence of borate is described. The method has been used as an assay for tRNA ribose methylases from yeast, using under methylated Escherichia coli tRNA as substrate. The main product formed with a partly purified yeast enzyme was characterized as 2'-O-methylcytidine.     

An embarrassment of riches: the enzymology of RNA modification

The maturation of transfer RNA (tRNA) involves extensive chemical modification of the constituent nucleosides and results in the introduction of significant chemical diversity to tRNA. Many of the pathways to these modified nucleosides are characterized by chemically complex transformations, some of which are unprecedented in other areas of biology. To illustrate the scope of the field, recent progress in understanding the enzymology leading to the formation of two distinct classes of modified nucleosides, the thiouridines and queuosine, a 7-deazaguanosine, is reviewed. In particular, recent data validating the involvement of several proposed intermediates in the formation of thiouridines are discussed, including two key enzyme intermediates and the activated tRNA intermediate. The discovery and mechanistic characterization of a new enzyme activity in the queuosine pathway is discussed.     

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV: 1H, 13C, and 15N NMR studies at natural isotope abundance.

The 1H, 13C, and 15N NMR spectra of neutral and protonated forms of the nucleosides 1-methyladenosine (m1A), 7-methylguanosine (m7G) and ethenoadenosine (EA), as a model compound, have been analyzed in order to assign the site of protonation in m1A and m7G. Protonation of these nucleosides occurs in the pyrimidine ring of m1A and EA and in the imidazole ring of m7G, with the charge being distributed rather than localized. Structural differences for both m1A and m7G were observed in solution and compared with those existing in the crystal state of monomers as well as in tRNA where these nucleosides occur quite often. The protonated nucleoside structures in solution compared favorably in sugar pucker and glycosidic bond conformations with x-ray crystallographic data. Methyl group carbon chemical shifts of the protonated mononucleosides corresponded to those of the methyls of the respective nucleosides in native tRNA structures. Therefore, the tRNA methyl group carbon chemical shifts are indicative of fully protonated nucleosides in the native, three dimensional structure of the nucleic acid.     

Nucleotide sequence of a lysine tRNA from Bacillus subtilis.

A lysine tRNA (tRNA1Lys) was purified from Bacillus subtilis W168 by a consecutive use of several column chromatographic systems. The nucleotide sequence was determined to be pG-A-G-C-C-A-U-U-A-G-C-U-C-A-G-U-D-G-G-D-A-G-A-G-C-A-U-C-U-G-A-C-U-U(U*)-U-U-K-A-psi-C-A-G-A-G-G-m7G(G)-U-C-G-A-A-G-G-T-psi-C-G-A-G-U-C-C-U-U-C-A-U-G-G-C-U-C-A-C-C-AOH, where K and U* are unidentified nucleosides. The nucleosides of U34 and m7G46 were partially substituted with U* and G, respectively. The binding ability of lysyl-tRNA1Lys to Escherichia coli ribosomes was stimulated with ApApA as well as ApApG.     

Temperature-dependent structural changes in complexes of tRNA(adenine-1)-methyltransferase from Thermus thermophilus HB8 and tRNA studied by optical and electron microscope methods

The complexation of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB8 (E.C.2.1.1.36) with Escherichia coli tRNA(Phe) and yeast tRNA1(Val) was investigated in a temperature range from 20 to 90 degrees C. The quantity of methylase subunits bounded with tRNA and the association constant (Ka) were determined by means of fluorescence quenching of the enzyme tryptophane residues by tRNA molecules. The number of enzyme subunits bounded with one tRNA molecule at temperatures 20-70 degrees C is equal to 8 +/- 2. The Ka values increase from (2 divided by 3).10(7) at 20 degrees C up to 8.5.10(7) M-1 at 70 degrees C. The temperature increase from 70 to 90 degrees C causes a decrease in the enzyme specific activity and in Ka values. In the temperature range from 75 to 90 degrees C a cooperative transition of methylase macromolecules into associates was observed. This association is accompanied by an increase of UV-light scattering and of fluorescence polarization coefficient of methylase tryptophane residues. In the absence of tRNA the size of enzyme associates (d) is evaluated to be more than 320 nm (d greater than or equal to lambda-320 nm), in the presence of tRNA-less than 320 nm (d much less than lambda-320 nm). An electron microscopic investigation of methylase and its complexes with tRNA at 20 degrees C revealed disk-like particles with a diameter and height of 8-11 nm and 4-5 nm, respectively. These disk-like methylase preparations dialized against distilled water form flexible polymeric rods with a diameter of 10-12 nm and the length of about several hundreds nm. During complexation of methylase with tRNA, in the same conditions as the dializes was carried out, large associates were not revealed.     

Number of genes and base composition of mitochondrial tRNA from Saccharomyces cerevisiae.

Increasing amounts of mitochondrial [32P] tRNA (4S fraction), were hybridized with mitochondrial DNA OF Saccharomyces cerevisiae. At saturation, the calculated number of genes for 4S mitochondrial RNA was 20. Mitochondrial [32P] tRNA eluted from the hydrids obtained either with an excess of tRNA or an excess of DNA showed, after alkaline hydrolysis and chromatography, a G+C content of 28 and 35 p. cent respectively. This last value is similar to that found with the total 4S fraction. The odd nucleotides T (about 1T per sequence), U, hU are present in mitochondrial tRNA. Some sequence may begin with pG.     

Complete analysis of tRNA-modified nucleosides by high-performance liquid chromatography: the 29 modified nucleosides of Salmonella typhimurium and Escherichia coli tRNA

A high-performance liquid chromatography (HPLC) method has been developed to quantify the major and modified nucleoside composition of total, unfractionated transfer RNA. The method is rapid and sensitive and offers a high degree of chromatographic resolution suitable for quantifying both stable and unstable modified nucleosides. It is nondestructive and allows the recovery of nucleosides for further characterization. We apply the method in the analysis of the 29 modified nucleosides in tRNA from Salmonella typhimurium (and Escherichia coli) and show it to be useful in examining changes in the modified nucleoside content of tRNA. Such changes may be important in regulation.     

The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNA(fMet)

Stepwise, solid-phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNA(fMet). The 34-ribonucleotide oligomer corresponding to the sequence of the 5'-half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis. This 34-nucleotide oligomer serves as an acceptor in the RNA-ligase-catalyzed reaction with a phosphorylated 43-ribonucleotide oligomer corresponding to the sequence of the 3'-half molecule of tRNA(fMet). The DNA molecule having the sequence of tRNA(fMet) is a 76-deoxyribonucleotide oligomer with a 3'-terminal riboadenosine residue and all U residues replaced by T. These polymers have been compared with an oligodeoxyribonucleotide lacking all 2'-hydroxyl groups except for the 3'-terminal 2'-OH, an oligoribonucleotide lacking modified nucleosides and E. coli tRNA(fMet). The all-RNA 77-nucleotide oligomer can be aminoacylated by E. coli methionyl-tRNA synthetase preparation from E. coli with methionine and threonylated in the A37 position using a yeast extract. In agreement with work by Khan and Roe using tDNA(Phe) and tDNA(Lys), the rA77-DNA(fMet) can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent. Kinetic data support the notion that aminoacylation of tRNA(fMet) does not depend on the presence of 2'-hydroxyl groups with the exception of that in the 3'-terminal nucleotide.