Aminoacylation of rat liver transfer RNA with homologous and heterologous enzyme systems during aging

Total tRNA extracted from livers of young (7 +/- 1 weeks), adult (40 +/- 1 weeks) and old (80 +/- 1 weeks) rats showed quantitative variation with age, being maximal in adults. Young and old animals yielded almost the same level of tRNAs. Quantitative changes in tRNAs were also observed from the study of amino acid acceptor activity using homologous enzyme i.e., aminoacyl-tRNA synthetase preparations from rat liver of the same age group. Quantitative variation followed the trend of qualitative variation. When tRNA was amino-acylated with a heterologous enzyme system, i.e., synthetase preparation from rat liver of another age group, age-related variation in aminoacyl-tRNA did not follow a pattern similar to that in the case of the homologous enzyme system. Young and adult synthetase enzymes showed maximum affinity for their homologous tRNAs but synthetases from old rat liver did not show any specific affinity for "old" tRNAs. This shows that apart from tRNAs, enzyme activity also changes with age.     

On dealing with anomalies in the transfer RNA aminoacylation reaction in partially purified systems

Apparent differences in tRNA and aminoacyl-tRNA synthetase complements in tissues undergoing differentiation have frequently been used to support theories of translational control. The validity of at least some of these studies must now be questioned because of anomalies in the tRNA aminoacylation reaction which can lead to incomplete aminoacylation of tRNA. Incomplete acylation of a tRNA mixture could result in different relative amounts of acylated isoaccepting species if acylation rates were not identical for all species. Using common methods of analysis, this situation could lead to misestimation of relative levels of isoacceptors or an inability to detect the presence of minor species. Bonnet and Ebel [Bonnet, J., and Ebel, J. (1972) Eur. J. Biochem.31, 335] used a highly purified valyl-tRNA and valyl-tRNA synthetase from yeast to demonstrate the presence of four reactions that occur simultaneously in that system. Herein, I apply the findings of Bonnet and Ebel to a mammalian system in a manner which is representative of attempts to study relative tissue proportions of tRNA isoacceptors. Total complements of tRNAs and the aminoacyl-tRNA synthetases have been partially purified from rabbit liver according to the methods of Yang and Novelli [Yang, W. K., and Novelli, G. D. (1971) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 20, p. 44, Academic Press, New York], probably the most commonly used procedures in the literature. Reaction conditions for tRNA acylation are shown to be modifiable so as to influence the extent of tRNA acylation. Procedures for optimizing the extent of tRNA acylation in such systems are demonstrated, and the unfavorable influence of Tris buffer, a factor not discussed by Bonnet and Ebel, is shown. Finally, examples of altered ratios of isoaccepting species in samples incompletely acylated due to suboptimal reaction conditions are provided.     

Aminoacylation of undermethylated mammalian transfer RNA.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.     

Valylation of the two RNA components of turnip-yellow mosaic virus and specificity of the tRNA aminoacylation reaction.

A comparative study of the aminoacylation of the two RNA components of turnip yellow mosaic virus, of yeast tRNAVal, tRNAfMet and of tRNAPhe by purified yeast valyl-tRNA synthetase is reported. Aminoacylations were performed in the presence of pure yeast tRNA nucleotidyltransferase, since 85% of the viral RNA molecules lacked the 3'-adenosine. We find that aminoacylation of the viral RNAs, like tRNA aminoacylation, reflects an equilibrium between the acylation and deacylation reactions. The kinetic parameters of TYM virus RNA valylation resemble the values found for tRNAVal valylation; in particular, there is a strong affinity between the viral RNA and valyl-tRNA synthetase and the rate constant for TYM virus RNA valylation is only slightly lower than that for tRNAVal. This result contrasts with the reduced rates observed in tRNA mischarging, and suggests that the viral RNA could be easily aminoacylated in vivo. Considering the fact that the 3'-terminal sequence of TYM virus RNA has only a few points of resemblance to a tRNA sequence, we propose that there are some structural motifs found in both tRNAVal and TYM virus RNA which are brought in a similar spatial arrangement recognized by valyl-tRNA synthetase.     

The RNA origin of transfer RNA aminoacylation and beyond

Aminoacylation of tRNA is an essential event in the translation system. Although in the modern system protein enzymes play the sole role in tRNA aminoacylation, in the primitive translation system RNA molecules could have catalysed aminoacylation onto tRNA or tRNA-like molecules. Even though such RNA enzymes so far are not identified from known organisms, in vitro selection has generated such RNA catalysts from a pool of random RNA sequences. Among them, a set of RNA sequences, referred to as flexizymes (Fxs), discovered in our laboratory are able to charge amino acids onto tRNAs. Significantly, Fxs allow us to charge a wide variety of amino acids, including those that are non-proteinogenic, onto tRNAs bearing any desired anticodons, and thus enable us to reprogramme the genetic code at our will. This article summarizes the evolutionary history of Fxs and also the most recent advances in manipulating a translation system by integration with Fxs.     

Aminoacylation of anticodon loop substituted yeast tyrosine transfer RNA

A procedure for replacing residues 33-35 in the anticodon loop of yeast tRNATyr with any desired oligonucleotide has been developed. The three residues were removed by partial ribonuclease A digestion. An oligonucleotide was inserted into the gap in four steps by using RNA ligase, polynucleotide kinase, and pseT 1 polynucleotide kinase. The rate of aminoacylation of anticodon loop substituted tRNATyr by yeast tyrosyl-tRNA synthetase was found to depend upon the sequence of the oligonucleotide inserted. This suggests that the nucleotides in the anticodon loop of yeast tRNATyr are required for optimal aminoacylation. In addition, tRNATyr modified to have a phenylalanine anticodon was shown to be misacylated by yeast phenylalanyl-tRNA synthetase at a rate at least 10 times faster than unmodified tRNATyr. Thus, the anticodon is used by phenylalanyl-tRNA synthetase to distinguish between tRNAs.     

Quantitative changes in aminoacylation of transfer RNA and in free amino acids during fructose-induced depletion of adenine nucleotides in rat liver

Fructose induces depletion of adenine nucleotides in liver and also strongly inhibits incorporation of radioactive amino acids into protein (Mäenpää, P.H., Raivio, K.O. and Kekomäki, M.P. (1968) Science 161, 1253–1254). In this study we have investigated the effects of fructose on aminoacylation of tRNA and on free amino acids in rat liver. 30 min after d-fructose (30 mmol/kg) was injected intraperitoneally into rats, liver ATP was reduced by 58%, ADP by 42%, AMP by 13%, the ATP/ADP ratio by 30%, and total adenine nucleotides by 48%. Using gas chromatography, the aminoacylation of tRNA was determined by quantifying the endogenous amino acids attached to tRNA in vivo. Aminoacylation was reduced by 31%. With different amino acids, reduction varied from 4% (asparagine plus aspartic acid) to 58% (arginine). On the other hand, the amount of free amino acids in the liver was increased by 24%. The most marked individual change was in alanine, which increased 5.7-times. This may have resulted from a combination of effects involving an increased production of alanine in muscle and liver and decreased hepatic gluconeogenesis from alanine caused by the ATP depletion.     

Chemical aminoacylation of transfer ribonucleic acid. The reaction of Escherichia coli tRNA with ethyl N-benzyloxycarbonylorthoglycinate.

The alpha-carbethoxypentadecyltrimethylammonium (Septonex) salt of tRNA (Ib) was condensed with ethyl N-benzyloxycarbonylorthoglycinate (II) in dimethylformamide in vacuo and in the presence of H3PO4 as catalyst. Pancreatic RNAase degradation and phenylalanine acceptor activity showed a 55--60% conversion to the 2',3'-cyclic orthoglycinate derivative of tRNA (IIIb). The orthoester grouping of IIIb was quantitatively hydrolyzed in 80% formic acid at 0 degrees C for 15 min to give 2'(3')-O-(N-benzyloxycarbonyl)glycyl tRNA (IVb). The latter was stripped at pH 8.8 to give tRNA whose behavior on DEAE cellulose column and gel electrophoresis was similar to that of starting tRNA. The phenylalanine acceptor activity amounted to almost 80% of the starting tRNA.     

Studies on the effects ofin vitro methyiation on aminoacylation of transfer RNA

In vitro methyiation ofEscherichia coli transfer ribonucleic acid by cell free extracts ofMycobacterium smegmatis leads exclusively to the formation of 1-methyl adenine [Vani, B. R., Ramakrishnan, T., Taya, Y., Noguchi, S., Yamaiuzumi, Z. and Nishimura, S. (1978)J. Bact., 137, 1085]. We have studied the effect of this modification on aminoacylationof Escherichia coli tRNA by mycobacterial enzymes. Aminoacylation with total algal protein hydrolysate as well as several individual aminoacids like methionine, valine, tyrosine, aspartic acid and lysine were monitored. In all the cases methyiation had a positive effect on the extent of aminoacylation by mycobacterial enzymes. Decreased aminoacylationin vitro was observed when hypomethylated transfer RNA from ethionine treated cells was used as the substrate for aminoacylation