Modification of guanine to queuine in transfer RNAs during development and aging.
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Aging↗
Biomedical subjects
N Shindo-Okada
Publications and source records attributed to N Shindo-Okada.
Biological and structural differences between tRNAVal species isolated from rat ascites hepatoma cells and normal rat liver.
On RPC-5 column chromatography, the main valine acceptor activity of tRNA (tRNA2Val) from rat ascites hepatoma cells was eluted later than that of normal rat liver tRNA (tRNA1Val). The tRNA2Val was aminoacylated by E. coli amino-acyl-tRNA synthetase, while tRNA1Val from normal rat liver was not. Rat fetal liver tRNAVal was also aminoacylated by E. coli aminoacyl-tRNA synthetase. tRNA1Val (rat liver) and tRNA2Val (ascites hepatoma) were each purified to a homogeneous state by RPC-5 column chromatography and two-dimensional polyacrylamide gel electrophoresis, and their sequences were determined by post-labeling techniques. Ascites hepatoma tRNA2Val differed from rat liver tRNA1Val in that Gm18, C32 and an unknown modified nucleoside, N34, in the latter tRNA were mostly replaced by G, Cm, and inosine, respectively. In addition, 3'-terminal adenosine was not present in tRNA1Val (normal rat liver), but was in tRNA2Val (ascites hepatoma). Other modifications and the primary structures of the two tRNAValS were found to be the same. Thus it was concluded that the new iso-acceptor species of tRNA Val in ascites hepatoma cells is due to a change of post-transcriptional modification, not to a change of tRNA transcription. The unique feature of the change of post-transcriptional modification in tRNA2Val (ascites hepatoma) is that both hypo- and hyper-modification take place simultaneously in the tRNA molecule depending the locations of nucleotide residues.
Amino Acids↗
Changes in amount of hypo-modified tRNA having guanine in place of queuine during erythroid differentiation of murine erythroleukemia cells.
The amounts of hypo-modified tRNAs having guanine in place of queuine in murine erythroleukemic cells decreased markedly when the cells differentiated into mature erythroid cells. The amounts of these hypo-modified tRNAs can be determined easily by measuring incorporation of labeled guanine into tRNA with Escherichia coli tRNA--guanine transglycosylase. The decrease was detected at on early stage of erythroid differentiation: namely, before any detectable increase in the percentage of cells containing hemoglobin. The amount of guanine-accepting tRNA species was nearly proportional to the percentage of undifferentiated cells in the population, regardless of the type of inducer used. Decrease in the amounts of hypo-modified tRNAs in the cells was effectively blocked by 12-O-tetradecanoylphorbol 13-acetate, which inhibits differentiation of these cells. 8-Azaguanine, which is known to be substrate of tRNA--guanine transglycosylase, was incorporated almost exclusively into the first position of hypo-modified tRNA in murine erythroleukemic cells when they were pulse-labeled in culture with 8-azaguanine, suggesting strongly that tRNA-guanine transglycosylase in the cells is actually involved in incorporation of 8-azaguanine into tRNA in vivo. The amount of 8-azaguanine incorporated into tRNA in differentiated cells was one third of that in undifferentiated cells, the decrease being parallel with that in the amount of guanine-accepting tRNA in these cells. The results suggest that the appearance of hypo-modified tRNAs in the transformed cells was due to lack of substrate for queuosine biosynthesis in tRNA.
Animals↗
Transfer ribonucleic acid guanine transglycosylase isolated from rat liver.
Transfer ribonucleic acid (tRNA) guanine transglycosylase (guanine insertion enzyme) was isolated from rat liver and extensively purified. The enzyme catalyzes an exchange of queuine (the base of queuosine, Q) as well as its precursors and guanine for guanine originally located in the first position of the anticodon of "undermodified" tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp from an Escherichia coli mutant or rat ascites hepatoma cells. This is in contrast to the previous observation that E. coli tRNA-guanine transglycosylase catalyzes the exchange of queuine precursors, such as 7-(aminoethyl)-7-deazaguanine and 7-cyano-7-deazaguanine, but not of queuine itself [Okada, N., Noguchi, S. Kasai, H., Shindo-Okada, N., Ohgi, T., Goto, T., & Nishimura, S. (1979) J. Biol. Chem. 254, 3067-3073]. The Km value for queuine of the rat liver enzyme is 9.2 X 10(-7) M, much lower than the values for the bases of queuosine precursors or guanine. Thus, the actual substrate for tRNA-guanine transglycosylase in queuosine biosynthesis in vivo in rat liver may not be 7-(aminomethyl)-7-deazaguanine, which is thought to be an actual substrate guanine, the E. coli system. Queuine or some queuine derivative may be the actual substrate for the tRNA-guanine transglycosylase reaction in the biosynthesis of Q in tRNA of mammalian cells. 6-Thioguanine and 8-azaguanine are also found to be good substrates.
Animals↗
Specific fluorescent labeling of 7-(aminomethyl)-7-deazaguanosine located in the anticodon of tRNATyr isolated from E. coli mutant.
Under-modified E. coli tRNATyr that contains 7-(aminomethyl)-7-deazaguanosine in place of Q nucleoside can be chemically modified by dansyl chloride under neutral conditions. Fluorescent labelling specifically occurred only in the 7-(aminomethyl)-7-deazaguanine moiety. The modified tRNATyr was found to be active both in aminoacylation and in binding to ribosomes.
Anticodon↗
Novel mechanism of post-transcriptional modification of tRNA. Insertion of bases of Q precursors into tRNA by a specific tRNA transglycosylase reaction.
The guanine insertion enzyme isolated from Escherichia coli (tRNA transglycosylase) catalyzed the incorporation of bases of Q (queuosine) precursors into E. coli undermodified tRNAAsn and tRNATyr. These bases of Q precursors were inserted in the first position of the anticodon of tRNASn and tRNATyr, replacing guanine originally located in that position. This is a novel type of post-transcriptional modification, inserting a modified base into the polynucleotide chain by cleavage of the N--C glycoside bond without breakage of the phosphodiester bond. One of the bases of Q precursors, 7-(aminomethyl)-7-deazaguanine, was found in the acid-soluble fraction of E. coli cells, supporting the conclusion that formation of Q, 7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine, in tRNA in vivo actually proceeds by the tRNA transglycosylase reaction.
Escherichia coli↗
Detection of unique tRNA species in tumor tissues by Escherichia coli guanine insertion enzyme.
The guanine insertion enzyme from Escherichia coli catalyzes exchange of guanine located at the first position of the anticodon of tRNA with radioactive guanine (N. Okada and S. Nishimura, unpublished data). tRNA isolated from various tumors, including slowly growing Morris hepatoma 7794A, incorporated considerable guanine with E. coli guanine insertion enzyme, whereas tRNA isolated from all normal tissues so far tested, except regenerating rat liver, incorporated scarcely any. In the rat ascites hepatoma AH7974, the guanine was mostly incorporated into minor isoaccepting species of tRNAAsp that contained the guanine residue instead of Q base in the first position of the anticodon. This is a sensitive and easy method for identifying unique tRNA species in tumor tissues.
Animals↗
Isolation of mammalian tRNAAsp and tRNATyr by lectin-Sepharose affinity column chromatography.
tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.
Animals↗
Rabbit liver tRNA1Val:I. Primary structure and unusual codon recognition.
The major valine acceptor tRNA1Val from rabbit liver was purified and its nucleotide sequence determined by in vitro [32P] - labeling with T4 phage induced polynucleotide kinase and finger-printing techniques. Its primary structure was found to be identical with the major valine tRNA from mouse myeloma cells. According to the wobble hypothesis this tRNA, which exclusively has an IAC anticodon, should decode the valine codons GUU, GUC and GUA only. However, this tRNA recognizes all four valine codons with a surprising preference for GUG. It is unknown whether this is due to the lack of A37 modification next to the 3' end of the anticodon IAC. The nature of the inosine-guanosine interaction remains to be clarified.
Animals↗