TRNA methylation

Enzyme ReguL 19, 427 (1980). Effect on polypeptide chain elongation in vitro A. K. Abraham, A. Pihl, Eur. J. Biochem 106, 257 (1980). Regulation of tRNA methyl transferase activity M. Mach et aL, Biochem J. 202, 153 (1982). HPLC study C. E. Prussak, D. H. Russell, J. Chromatog. 229, 47 (1982). Interaction with actin, q.v. C. Oriol-Audit, Biochem. Biophys. Res. Commun. 105, 1096 (1982). Studies on use as a biochemical tool in career research A. Thyss et L, Eur. J- Cancer Clin, OncoL 18, 611 (1982) V, Quemener... 

Isomerizations, e. g. methylmalonyl-CoA to succinyl- Vitamin B,2 CoA. Also ribonucleotide triphosphate reduction, homocysteine methylation, tRNA methylation, methane production by methanogenic bacteria, etc. 

As it was shown also by the data reported above, tRNA methyl-transferases exibit a very complex kind of specificity toward the tRNA substrate. In fact, three requirements must be fulfilled in order to achieve the enzymatic attachment of a methyl group to tRNA. Each enzyme must recognize (i) the proper moiety along the polynucleotide chain (either the specific base of the ribose) (ii) the position of the modification at the purine, pyrimidine or furanosic rings (Hi) the localized nucleotide sequence and the spatial locus of the three-dimensional configuration in which the methyl-atable nucleoside is positioned. These three requirements allow us to define three different types of specificity for tRNA methyltrans-ferase, namely moiety specificity, ring-atom specificity and site specificity, respectively. 

Selenocysteine was identified in 1976 (57) in a protein produced by Clostridium stricklandii, and it is thought to be the form in which selenium is incorporated, stoichiometricaHy, into proteins. Studies with rats show that over 80% of the dietary selenium given them is incorporated into proteins, thus selenocysteine takes on metaboHc importance. Selenoproteins having known enzymatic activities contain selenocysteine at the active sites. Two other forms of metabohc selenium are recognized methylated selenium compounds are synthesized for excretion, and selenium is incorporated into some transfer ribonucleic acids (tRNAs) in cultured cells (58). Some of the more important seleno-compounds are Hsted in Table 4. Examples of simple ring compounds are shown in Eigure 4. 

In recent year s, clinical studies on the role of uiinai y luodified nucleosides as the biochemical mai kers of various types of cancer have been actively undertaken. Most of the urinai y modified nucleosides ai e piimai ily originated by methylation of either the base part, the sugar hydroxyl par t, or in some cases, both par ts of the course of biodegradation of tRNA molecules. Hence, their isolation and identification plays a major role in biochemical analysis. 

Transfer RNAs normally contain some bases other than A, U, G, and C. Of the 76 bases in tRNA , for example, 13 are of the modified variety. One of these, marked G in Figure 28.11, is a modified guanosine in the anticodon. Many of the modified bases, including G, are methylated derivatives of the customary RNA bases. 

Transfer RNA (tRNA) serves as a carrier of amino acid residues for protein synthesis. Transfer RNA molecules also fold into a characteristic secondary structure (marginal figure). The amino acid is attached as an aminoacyl ester to the 3 -terminus of the tRNA. Aminoacyl-tRNAs are the substrates for protein biosynthesis. The tRNAs are the smallest RNAs (size range—23 to 30 kD) and contain 73 to 94 residues, a substantial number of which are methylated or otherwise unusually modified. Transfer RNA derives its name from its role as the carrier of amino acids during the process of protein synthesis (see Chapters 32 and 33). Each of the 20 amino acids of proteins has at least one unique tRNA species dedicated to chauffeuring its delivery to ribosomes for insertion into growing polypeptide chains, and some amino acids are served by several tRNAs. For example, five different tRNAs act in the transfer of leucine into... 

Posttranslational modification of preformed polynucleotides can generate additional bases such as pseudouridine, in which D-ribose is linked to C-5 of uracil by a carbon-to-carbon bond rather than by a P-N-glycosidic bond. The nucleotide pseudouridylic acid T arises by rearrangement of UMP of a preformed tRNA. Similarly, methylation by S-adenosylmethionine of a UMP of preformed tRNA forms TMP (thymidine monophosphate), which contains ribose rather than de-oxyribose. 

FormylMet-tRNA Formate I < S-Formylglutathione Formaldehyde methyl amino acids... 

Using phosphotriester methods, dinucleoside (3 - 50-monophosphates containing 6-methyl-2,-deoxyuridine at the 3 - or 5 -end have been prepared.44 N.m.r. spectroscopy indicates that this nucleoside possesses the syn conformation in these compounds, and, on treatment with snake venom phosphodiesterase, d(m6UpT) is degraded, while d(Apm6U) is not, indicating that this enzyme, a 3 -exonuclease, requires the anti conformation to be present in the substrate. Two modified nucleo-side-5 -monophosphates, (20) and (21), which are resistant to 5 -nucleotidase, have been isolated from tRNA snake venom hydrolysates.45 A synthesis of (20) has been reported.46... 

GatCAB amidotransferase.This natural product mimics the charged 3 -terminus of aa-tRNA and has been used as a tool for the study of protein biosynthesis. The parent compound 22 is a very weak inhibitor of AdT. The amino acid chain is related to tyrosine and differs from the glutamic and aspartic side chains transformed in the kinase or the transamidase steps. Replacement of the methoxyphenyl moiety of puromycin by carboxylic acid derivatives (23-26) improved the ability to inhibit this AdT. Stable analogues of the transition state in the last step of the transamidation process (27-29) where the carbonyl to be attacked by NH3 is replaced by tetrahedral sulfur or phosphorus atom with a methyl group mimicking ammonia exhibited the highest activity. 

Figure 12.5 The structures for four tRNA molecules of yeast, (a) Alanyl-tRNA (b) phenylalanyl-tRNA (c) seryl-tRNA (d) tyrosyl-tRNA. The single letter designations identify the sequence of bases along the single chain. Note that several of these are unusual bases, most of which are methylated (Me). Note also the ACC sequence at the 3 terminus of each tRNA. This is the site to which amino acids are attached in the process of protein synthesis, as indicated. These tRNA molecules have a substantial amount of secondary structure created by formation of Watson-Crick base pairs. Finally, note that the anticoding triplet in the bottom loop is shown.

The bases that occur in nucleic acids are aromatic heterocyclic compounds derived from either pyrimidine or purine. Five of these bases are the main components of nucleic acids in all living creatures. The purine bases adenine (abbreviation Ade, not A ) and guanine (Gua) and the pyrimidine base cytosine (Cyt) are present in both RNA and DNA. In contrast, uracil (Ura) is only found in RNA. In DNA, uracil is replaced by thymine (Thy), the 5-methyl derivative of uracil. 5-methylcyto-sine also occurs in small amounts in the DNA of the higher animals. A large number of other modified bases occur in tRNA (see p. 82) and in other types of RNA. 

The base sequence and the tertiary structure of the yeast tRNA specific for phenylalanine (tRNA " ) is typical of all tRNAs. The molecule (see also p.86) contains a high proportion of unusual and modified components (shaded in dark green in Fig. 1). These include pseudouridine (T), dihydrouridine (D), thymidine (T), which otherwise only occurs in DNA, and many methylated nucleotides such as 7-methylguanidine (m G) and—in the anticodon—2 -0-methylguanidine (m G). Numerous base pairs, sometimes deviating from the usual pattern, stabilize the molecule s conformation (2). 

Methyltransferases that utilize S-adenosyl-L-methionine as the methyl donor (and thus generating S-adenosyl-L-homocysteine) catalyze (a) A-methylation (e.g., norepinephrine methyltransferase, histamine methyltransferase, glycine methyltransferase, and DNA-(adenine-A ) methyltransferase), (b) O-methylation (e.g., acetylsero-tonin methyltransferase, catechol methyltransferase, and tRNA-(guanosine-0 ) methyltransferase), (c) S-methyl-ation (e.g., thiopurine methyltransferase and methionine S-methyltransferase), (d) C-methylation (eg., DNA-(cy-tosine-5) methyltransferase and indolepyruvate methyltransferase), and even (e) Co(II)-methylation during the course of the reaction catalyzed by methionine syn-thase. ... 

The final type of tRNA processing is the modification of some of the bases by methylation, deamination, or reduction (Fig. 26-24). In the case of pseudouridine Q ), the base (uracil) is removed and reattached to the sugar through C-5. Some of these modified bases occur at characteristic positions in all tRNAs (Fig. 26-23). 

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