Occurrence of 1-methyladenosine and absence of ribothymidine in transfer ribonucleic acid of Mycobacterium smegmatis.

The minor base composition of Mycobacterium smegmatis tRNA has been studied. Thin-layer chromatographic patterns of a ribonuclease T2 digest of mycobacterial tRNA indicated the presence of appreciable amounts of 1-methyladenosine (which is commonly present only in eucaryotic tRNA), dihydrouridine, and 7-methylguanosine. Ribothymidine was absent. The S-adenosylmethionine-dependent tRNA methylases of M. smegmatis catalyzed the formation of 1-methyladenosine when Escherichia coli tRNA was used as acceptor. Similarly, E. coli extracts methylated the tRNA of M. smegmatis, forming ribothymidine.     

N2-guanine specific transfer RNA methyltransferase I from rat liver and leukemic rat spleen

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N(2)-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m(2)G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA(Arg), tRNA(Phe), and tRNA(Val) yielded significantly more m(2)G than the bulk tRNA. The K(m) for tRNA(Arg) in the methylation reaction with enzymes from either tissue was 7.8 x 10(-7) M as compared to the value 1 x 10(-5) M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA(Arg), tRNA(Phe), and tRNA(Val), A-m(2)G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5'-end contained in a sequence (s(4))U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Utilization of an Escherichia coli mutant for carbon-13 enrichment of tRNA for NMR studies.

The enrichment of tRNA at specific sites with carbon-13 has been accomplished in vivo using a mutant of Escherichia coli. A relaxed strain of E. coli auxotrophic for methionine was grown in a specifically defined medium supplemented with either [14C] or [13C]-methyl labeled methionine. Cells were collected at the end of the log-phase of growth and tRNA was extracted. Analysis of the radioactivity of the [14C]-labeled tRNA established an incorporation ratio of three labeled carbons per tRNA molecule. Incorporation of the [14C]-label in vivo was confined to the methylation of nucleotides as determined by thin layer chromatography of nucleotides resulting from a ribonuclease digestion of [14C]-labeled tRNA. The carbon-13 NMR spectrum of [13C]-enriched tRNA indicated a similar degree of incorporation into the methylated nucleotides by the substantial enhancement of [13C]-methyl NMR signals only. Assignment of signals has been made for the methyl groups of ribothymidine and N7-methylguanosine in E. coli tRNA.     

Isolation and partial characterization of three Escherichia coli mutants with altered transfer ribonucleic acid methylases.

Seven transfer ribonucleic acid (tRNA) methylase mutants were isolated from Escherichia coli K-12 by examining the ability of RNA prepared from clones of unselected mutagenized cells to accept methyl groups from S-adenosylmethionine catalyzed by crude enzymes from wild-type cells. Five of the mutants had an altered uracil-tRNA methylase; consequently their tRNA's lacked ribothymidine. One mutant had tRNA deficient in 7-methylguanosine, and one mutant contained tRNA lacking 2-thio-5-methylaminomethyluridine. The genetic loci of the three tRNA methylase mutants were distributed over the E. coli genome. The mutant strain deficient in 7-methylguanosine biosynthesis showed a reduced efficiency in the suppression of amber mutations carried by T4 or lambda phages.     

Transfer RNA methyltransferase activity in paramecium aurelia.

The tRNA methyltransferases from Paramecium aurelia were investigated. The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C. The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of tRNA in P. aurelia is similar to that observed in other eukaryotes.     

1H NMR of valine tRNA modified bases. Evidence for multiple conformations.

Methyl and methylene protons of dihydrouridine 17 (hU), 6-methyladenosine 37 (M6A), 7-methylguanosine 46 (m7G), and ribothymidine 54 (rT) give clearly resolved peaks (220 MHz) for tRNA1val (coli solutions in D2O, 0.25 m NaCl, at 27 degrees C. Chemical shifts are generally consistent with a solution structure of tRNA1val similar to the crystal structure of tRNAphe (yeast). At least 3 separate transitions are observed as the temperature is raised. The earliest involves disruption of native tertiary structure and formation of intermediate structures in the m7G and rT regions. A second transition results in a change in structure of the anticodon loop, containing m6A. The final step involves unfolding of the m7G and rT intermediates and melting of the TpsiC helix. Low salt concentrations produce multiple, partially denatured conformations, rather than a unique form, for tRNA1val. Native structure is almost completely reformed by addition of Na+ but Mg2+ is required for correct conformation in the vicinity of m7G.     

Regulation of tRNA methyltransferase activities by spermidine and putrescine. Inhibition of polyamine synthesis and tRNA methylation by alpha-methylornithine or 1,3-diaminopropan-2-ol in Dictyostelium.

Inhibitors of polyamine synthesis (alpha-methylornithine and 1,3-diaminopropan-2-ol) were used to study the relationship between polyamine synthesis and specific methylations of tRNA in Dictyostelium discoideum during vegetative growth. Polyamine concentrations were found to be 10 mM for putrescine, 1.6 mM for spermidine and 7 mM for 1,3-diaminopropane throughout the growth stage. On treatment of growing amoebae with alpha-methylornithine or with 1,3-diaminopropan-2-ol (each at 5 mM), the syntheses of putrescine, spermidine and 1,3-diaminopropane were arrested within 4h. After polyamine synthesis had ceased, the incorporation of methyl groups into tRNA was considerably decreased under conditions that had no effect on the incorporation of uridine into tRNA, or on net syntheses of protein and of DNA. The following nucleosides in tRNA were concerned: 1 methyladenosine, 5-methylcytidine, 7-methylguanosine, 2-methylguanosine, N2N2-dimethylguanosine and 5-methyluridine (ribosylthymine). The corresponding tRNA methyltransferases, determined in Mg2+-free enzyme extracts, proved to be inactive unless polyamines were added. Putrescine and/or spermidine at concentrations of 10 mM or 1-2 mM respectively stimulate the transmethylation reaction in vitro to a maximal rate and to an optimal extent at exactly the same concentrations as found in vegetative cells. In contrast, 1,3-diaminopropane, which is formed from spermidine, does not affect the methylation of tRNA in vitro at physiological concentrations. Putrescine and/or spermidine stabilize the tRNA methyltransferases in crude extracts in the presence but not in the absence of the substrate tRNA. The results support the view that S-adenosylmethionine-dependent transmethylation reactions can be regulated by alterations of polyamine concentrations in vivo.     

Formylatable methionine transfer RNA from Mycoplasma: purification and comparison of partial nucleotide sequences with those of other prokaryotic initiator tRNAs.

The major species of the formylatable methionine tRNA from Mycoplasma mycoides var capri has been purified. The 5'- and 3'-terminal sequences of the purified tRNA are pC-G- and C-A-A-C-C-AOH, respectively. Thus, this tRNA also contains the unique structural feature found in two other prokaryotic initiator tRNAs in that the first nucleotide at the 5'-end cannot form a Watson-Crick type of base-pair to the fifth nucleotide from the 3'-end. The Mycoplasma tRNA does not contain ribothymidine; however, a specific uridine residue in the sequence G-U-psi-C-G- can be enzymatically methylated by E. coli extracts to yield G-T-psi-C-G. Since ribothymidine is absent in crude tRNA from this strain of Mycoplasma, the absence of T is probably due to the lack of a U yields T modifying enzyme.     

Initiation of Protein Synthesis Without Formylation in a Mutant of Escherichia coli That Grows in the Absence of Tetrahydrofolate

Starting from a p-aminobenzoate-requiring strain of Escherichia coli (E. coli K-12 AB3292), we have isolated mutants that can grow in the absence of p-aminobenzoate (and thus tetrahydrofolate). The following lines of evidence suggest that at least one of these mutants is capable of initiating protein synthesis without formylation of methionyl-transfer ribonucleic acid (methionyl-tRNA(fMet)). (i) tRNA isolated (and charged in vivo with [(35)S]methionine) from this mutant grown in a p-aminobenzoate-free medium contained less than 0.4% of the total methionine charged to the tRNA as formylmethionine. However, when the mutant was grown in the presence of p-aminobenzoate, 40 to 50% of the total [(35)S]methionine was detected as formylmethionine. (ii) Extracts of the mutant grown in the absence of p-aminobenzoate contained no formyl-tetrahydrofolate, but such extracts did contain formylatable methionyl-tRNA and a functional transformylase. (iii) Tetrahydrofolate-free extracts of the mutant were capable of supporting protein synthesis with viral RNA (from f2) as messenger, but the resulting synthesized proteins contained no formylmethionine, and methionine residues were detected where formylmethionine residues are normally found. In the presence of formyl-tetrahydrofolate, use of a similar extract resulted in the detection of 30 to 40% of the total polypeptide methionine as formylmethionine. (iv) Initiation of protein synthesis in vitro occurred more readily with formyl-tetrahydrofolate-free extracts of the mutant than with similar extracts prepared from the parent strain. However, in the presence of formyl-tetrahydrofolate, initiation of protein synthesis proceeded equally well with both kinds of extracts. tRNA from this mutant and another spontaneously derived mutant was found to be partially deficient in the modified nucleoside ribothymidine (rT). Analysis of extracts showed that the mutants contained decreased levels of the methylase that results in the formation of ribothymidine. In vivo studies with an independently isolated rT(-) strain suggest that the lack of rT in tRNA facilitates the growth of E. coli under conditions where protein synthesis is forced to take place without formylation.     

The yggH gene of Escherichia coli encodes a tRNA (m7G46) methyltransferase

We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-L-methionine-dependent formation of N(7)-methylguanosine at position 46 (m(7)G46) in tRNA. Additionally, we generated an E. coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m(7)G46) methyltransferase activity.     

Wheat germ cytoplasmic ribosomes. Localization of 7-methylguanosine and 6-methyladenosine by electron microscopy of immune complexes

Nucleoside analysis of the RNA from the small subunit of wheat germ cytoplasmic ribosomes shows 1 mol each of N7-methylguanosine and N6-methyladenosine/mol of RNA. Antibodies directed against each methylated nucleoside were used to localize these residues within the subunit by electron microscopy of immune complexes. Antibodies to 7-methylguanosine bound 40 S subunits at a single site, at or slightly above the division between the upper and lower segments of the particle and on the surface furthest from the platform (or large lobe) of the subunit. This site is essentially equivalent to that previously seen with Escherichia coli and chloroplast 30 S subunits (Trempe, M. R., Ohgi, K., and Glitz, D. G. (1982) J. Biol. Chem. 257, 9822-9829). Antibodies to N6-monomethyladenosine were induced in rabbits with a nucleoside-albumin conjugate and shown to be specific for the modified nucleoside. Electron microscopy of antibody-subunit complexes placed the methyladenosine residue in a position that is essentially indistinguishable from that of 7-methylguanosine.     

Transfer RNA methyltransferases from yellow lupin seeds: purification and properties.

tRNA methyltransferases from extract of yellow lupin seeds were purified over 300-fold by the methods based on hydrophobic and affinity chromatography. However, in the most active fractions the methylating enzymes were over 2000 purified. The purified enzyme fractions catalysed the formation of 1-methyladenine and 5-methylcytosine using E. coli B and B. subtilis tRNAs as substrates and S-adenosylmethionine as the methyl donor. They were unable to methylate their own endogenous tRNA but they were capable of methylating tRNA of some other lupinus species. Whereas the patterns of methylated constituents of tRNA of some other lupinus and B. subtilis were quite similar, they differed considerably from those obtained with lupin species tRNAs. Some properties of purified methyltransferases from yellow lupin seeds have been described.     

Purine N7 groups that are crucial to the interaction of Escherichia coli rnase P RNA with tRNA.

We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+). The majority of identified positions represent highly or universally conserved nucleotides. Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306). Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA-RNase P RNA conjugate in the presence of 3 M NH4+ or Na+. This indicates that these N7 functional groups play a key role in the structural organization of ribozyme-substrate and -product complexes. We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E. coli RNase P RNA. This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E. coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76).     

Studies on lysine, glutamine and glutamic acid tRNAs from Drosophila.

The minor bases present in the family of Drosophila tRNAs recognising codons of the type NAA or NAG have been studied. Under standard aminoacylating conditions, the acceptor activities of BrCN-treated tRNA-Lys-5 tRNA-Glu-4 and tRNA-G1n-4 were completely eliminated, suggesting the presence of 2-thiouridine derivatives. The two major lysine tRNA species (tRNA-Lys-2 and tRNA-Lys-5) were purified and their nucleoside content determined both directly and by the tritium derivative technique. Both tRNAs contain 1-methyladenosine, N-2-dimethylguanosine, 7-methylguanosine, 5-methylcytidine, pseudouridine and dihydrouridine, and tRNA-Lys-5 contains 1-methylguanosine. Neither species contain ribothymidine, although both may contain 2'-O-methyl ribothymidine. A nucleoside with ultraviolet spectral properties similar to N-4-acetylcytidine was found in tRNA-Lys-5 and a nucleoside with chromatographic properties the same as N-[9-beta-D-ribofuranosyl)-purin-6-yl-carbamoyl] threonine was found in tRNA-Lys-2. A 2-thiouridine derivative was not found in tRNA-Lys-5 using these chromatographic techniques.     

Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine

Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids [( 75Se]tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32-. When E. coli [75Se]tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA. This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-[(methylamino)methyl]-2-selenouridine. Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties. The new selenonucleoside also accounted for 40-60% of the 75Se found in [75Se]tRNA from C. sticklandii or M. vannielii. Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-[(methylamino)methyl]-2-selenouridine. Pure seleno-tRNAGlu isolated from C. sticklandii contains one 5-[(methylamino)methyl]-2-selenouridine and one 4-thiouridine per tRNA molecule.     

Tetrahydrofolate-dependent biosynthesis of ribothymidine in transfer ribonucleic acids of Gram-positive bacteria.

Trimethoprim, an inhibitor that prevents tetrahydrofolate-dependent transmethylation reactions inbacteria, was used in a comparative study to discriminate between two possible biosynthetic pathways, either the S-adenosylmethionine or the tetrahydrofolate-dependent formation of ribothymidine (rT) in transfer ribonucleic acids (tRNA's) of several strains of gram-positive and gram-negative microorganisms. rT-deficient tRNA's accumulate in trimethoprim-treated gram-positive Streptococcus faecium, Staphylococcus aureus, Corynebacterium bovis, Arthrobacter albidus, and all examined Bacillaceae, except Bacillus stearothermophilus. The rT-deficient rT-deficient tRNA's accept the methyl moiety from S-adenosylmethionine in vitro, with extracts from Escherichia coli (wild type) as a source of methylating enzymes; 90% of the incorporated methyl groups are present in rT. Trimethoprim does not inhibit the biosynthesis of rT in tRNA of gram-negative Enterobacteriaceae, Rhizobium lupini, and Pseudomonadaceae, suggesting that the rT-specific tRNA methyltransferases of these gram-negative strains use S-adenosylmethionine as coenzyme.     

Origin and formation of 1,7-dimethylguanosine in tRNA chemical and enzymatic methylation

tRNA chemical methylation: 1. 1,7-Dimethylguanosine was found in in vivo methylated tRNA from liver and kidney of rat after exposure to a low dose of dimethylnitrosamine (4 mg/kg body weight). 2. At 4 h after dimethylnitrosamine administration, the 1,7-dimethylguanosine:7-methylguanine ratio (product ratio) for liver and kidney tRNA was 0.017 and 0.091, respectively. At 24 h after dimethylnitrosamine administration, the product ratio was lower in both hepatic and renal tRNA. 3. When dimethylnitrosamine was given in four separate daily injections, the product ratio in hepatic tRNA 4 h after the last dose was the same as for the same total dose given by a single injection, but in renal tRNA it was lower. No dialkyl compound was found in liver and kidney tRNA 24 h after the last multiple injection. tRNA enzymatic methylation: 1. Base analyses of Escherichia coli B tRNA methylated in vitro, by using S-adenosylmethionine as physiological methyl donor and enzyme preparations from liver and kidney of normal rat, indicated that 1,7-dimethylguanosine was also a product of enzymatic methylation. 2. The amount of 1,7-dimethylguanosine formed by kidney enzyme preparation was 3-times that produced by the liver extract. 3. A second type of enzymatic methylation assay where chemically methylated tRNA was used as substrate indicated that the 7-methylguanosine residues in the nucleic acid are not the substrate of the methylase activity forming the 1,7-dimethylguanosine moieties. Analogous data were obtained for the origin of 1,7-dimethylguanosine residues in tRNA chemical methylation by dimethyl sulphate.     

Modified nucleosides of Bacillus subtilis transfer ribonucleic acids.

An analysis of the kinds and amounts of minor nucleosides of transfer ribonucleic acids (tRNA's) from Bacillus subtilis 168 trpC2 is presented. Identification and quantitation were accomplished using ion exclusion chromatography, thin-layer and paper chromatography, and ultraviolet absorption properties. Nucleosides and their amount in moles per 80 residues are as follows: guanosine (25.7), cytidine (22.0), adenosine (15.2), uridine (13.1), 5-methyluridine (0.98), pseudouridine (1.54), 1-methyladenosine (0.15), N6-methyladenosine (0.01), 7-methyladenosine (0.10), 2-methyladenosine (0.03), 7-methylguanosine (0.20), N2-methylguanosine (0.14), 1-methylguanosine (0.14), a methylated pyrimidine (0.17), a methylated derivative of N6-(delta 2-isopentenyl)adenosine (0.02), ribose methylated nucleosides (0.02), 4-thiouridine (0.12), 2-thio-5-(N-methylaminomethyl) (0.09), and an unknown thionucleoside (0.12). Although the composition is similar to that of Escherichia coli in the proportion of major nucleosides, the content of pseudouridine and 5-methyluridine, and the degree of base and ribose methylation, the composition is more similar to that of the tRNA's of yeast and higher organisms in its lower degree of thiolation, the presence of significant amounts of 1-methyladenosine, and the low levels of 2-methyladenosine and 6-methyladenosine. Therefore, the nucleoside composition of B. subtilis presents some different aspects from those usually given as characteristic for bacterial tRNA's. It is not known whether these differences are due to variation between bacterial species in general or related to the process of differentiation.     

On the substrate specificity of DNA methyltransferases. adenine-N6 DNA methyltransferases also modify cytosine residues at position N4

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methylated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M. FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines. This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base. This unexpected finding has implications on the mechanism of adenine-N6 DNA methyltransferases. Sequence comparisons suggest that adenine-N6 and cytosine-N4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family.